EP3143392A1 - Quantitation of tamoxifen and metabolites thereof by mass spectrometry - Google Patents

Abstract

Provided are methods for determining the amount of tamoxifen and its metabolites in a sample by mass spectrometry. In some aspects, the methods provided herein comprise determining the amount of norendoxifen. In some aspects, the methods provided herein comprise determining the amount of norendoxifen and tamoxifen. In some aspects, the methods provided herein comprise determining the amount of norendoxifen and other tamoxifen metabolites. In some aspects, the methods provided herein comprise determining the amount of tamoxifen, norendoxifen, and other tamoxifen metabolites.

Description

QUANTITATION OF TAMOXIFEN AND METABOLITES THEREOF BY MASS

SPECTROMETRY

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS [8001] This application claims benefit of U.S. Provisional Application No. 61/992,214, filed May 12, 2014, which is incorporated by reference herein in its entirety.

BACKGROUND OF THE IN VENTION

[8002] The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.

[0003] Tamoxifen is a standard of treatment for hormone receptor positive breast cancer patients after primary treatment. Five to ten years of tamoxifen therapy reduces the risk of recurrence and death in these patients. Yet, many patients do not complete their full course of therapy, frequently due to unpleasan t side effec ts of the drug.

[0004] Tamoxifen is a pro-drag which is converted to the highly active Endoxifen for full effectiveness. Conversion of Tamoxifen to Endoxifen is through a metabolic pathway dependent on genetic variation, such as that in CYP2D6 (2D6). Although 2D6 genotyping has been promoted to predict response to Tamoxifen and toxicity, the direct association on an individual level is controversial.

[8005] An effective method of predicting response to Tamoxifen is needed.

SUMMARY OF THE INVENTION

[8006] The present invention provides methods for quantitation of tamoxifen and its metabolites in a sample by mass spectrometry, including tandem mass spectrometry.

[0007] In one aspect, methods are provided for determining the amount of norendoxifen in a sample by mass spectrometry, comprising (a) ionizing said norendoxifen to produce one or more norendoxifen ions detectable by mass spectrometry; (b) detecting the amount of the norendoxifen ion(s) from step by mass spectrometry; wherein the amount of the ion(s) detected is related to the amount of norendoxifen in said sample.

800 ] In one aspect, methods are provided for determining the amount of tamoxifen and metabolites thereof in a sample in a single mass spectrometry assay, comprising (a) ionizing said tamoxifen and metabolites to produce one or more ions detectable by mass spectrometry; (b) detecting the amount of the ion(s) from step by mass spectrometry; wherein the amount of the ion(s) detected is related to the amount of each of tamoxifen and metabolites in said sample.

000 ] In some embodiments, said metabolites comprise norendoxifen. In some embodiments, said metabolites comprise endoxifen or N-Desmethyl-4-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise 4 '-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise 4 -Hydroxy Tamoxifen. In some embodiments, said metabolites comprise N-Desmethy 1-4' -Hydroxy Tamoxifen. In some embodiments, said metabolites comprise N-Desmeihyi Tamoxifen. In some embodiments, said metabolites comprise metabolites selected from the group consisting of norendoxifen, endoxifen, 4'- Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N-Desmethyl-4'-Hydroxy Tamoxifen, and N- Desmethyl-4'-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise any combination of norendoxifen, endoxifen, 4'-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N - Desmethy 1-4' -Hydroxy Tamoxifen, and N-Desmethyl Tamoxifen. In some embodiments, said metabolites comprise norendoxifen, endoxifen, 4'-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N-Desmethyl-4'-Hydroxy Tamoxifen, and N-Desmethyl-4'-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise any combination of norendoxifen, endoxifen, 4 '-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, -Desmethyl-4' -Hydroxy Tamoxifen, and N-Desmethyl Tamoxifen.

[0010] In one aspect, methods provided herein comprise protein precipitation. In some embodiments, methods provided herein comprise purification. In some embodiments, said purification comprises filtration. In some embodiments, said purification comprises liquid chromatography. In some embodiments, said liquid chromatography is high pressure liquid chrom tography (HPLC) .

[8011] In some embodiments, methods provided herein comprise detecting the amount of an internal standard. In some embodiments, said internal standard is a deuterated norendoxifen. [8(512] In some embodiments, ionization is by atmospheric pressure chemical ionization (APCI), In some embodiments, said ionization is in positive ion mode.

[8013] In some embodiments, ionization is by electrospray ionization (ESI). In some embodiments, said ionization is in positive ion mode.

[8014] In some embodiments, said sample is a serum sample.

[8815] In some embodiments, mass spectrometry is tandem mass spectrometry.

[0016] In one aspect, provided herein is a method for predicting tamoxifen response in a patient by determining the amount of tamoxifen or one or more tamoxifen metabolites. In some embodiments, a high amount of one or more tamoxifen or tamoxifen metabolites indicate a positive response to tamoxifen in a patient. In some embodiments, said metabolites comprise norendoxifen. In some embodiments, said metabolites comprise endoxifen or N- Desmethyl-4-Hydroxy Tamoxifen. In some embodiments, said metabolites comprise 4'- Hydroxy Tamoxifen. In some embodiments, said metabolites comprise 4-Hydroxy

Tamoxifen. In some embodiments, said metabolites comprise -Desmethyl-4' -Hydroxy Tamoxifen. In some embodiments, said metabolites comprise N-Desmethyl Tamoxifen. In some embodiments, said metabolites comprise norendoxifen, endoxifen, 4'-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N-Desmethyl-4'-Hydroxy Tamoxifen, and N-Desmethyl- 4 ' -Hydroxy Tamoxifen.

[8817] In some embodiments, the method provided herein has sensitivity measured by limit of quantitation (LOQ). In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 4 ng/mL, In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 3 ng/mL, in some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL.

[8018] In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiment s, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl T amoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL.

[8(519] In some embodiments, the method of quantitation of 4'-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of 4' -Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of 4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of 4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of 4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of 4' -Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.4 ng/mL. In some embodiments, the method of quantitation of 4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.2 ng/mL.

8028] In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantit ation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.4 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.2 ng/mL.

[8021] In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of -Desmethyl-4-Hydroxy Tamoxifen

(Endoxifen) has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of N-DesmethyI-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments; the method of quantitation of -Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desm.ethyl-4- Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4- Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 1.5 ng/mL. In some

embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen

(Endoxifen) has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of -Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 0.4 ng/mL.

[8022] In some embodiments, the method of quantitation of N-Desmethyl-4'-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitaiion of N -Desmethyl-4' -Hydroxy Tamoxifen has a limit of quantitaiion less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of N- Desmethyl-4' -Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4'-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyi-4'-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of N-Desmeth l-4'- Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4'-HydiOxy Tamoxifen has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of N-Desmetbyl-4'- Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.4 ng/mL.

[8023] In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 3 ng/mL. m some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 1.5 ng mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 1.2 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 1 ng mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 0.5 ng/mL.

[8024] In some embodiments, the method provided herein has sensitivity measured by limit of detection (LOD). In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detec tion less than or equal to 0.6 ng/mL.

[8025] In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of -Desmethyi. ^'T amoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N- Desmethyl Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmetbyl Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of N- Desmethyl Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, ihe method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of N- Desmethyl Tamoxifen has a limit of detection less than or equal to 0.6 ng/mL.

[0026] In some embodiments, the method of detection of 4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of 4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of 4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of 4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of 4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of 4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of '-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.4 ng/mL. In some embodiments, the method of detection of 4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.2 ng/mL, In some embodiments, the method of detection of 4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.1 ng/mL.

[8027] In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the me thod of detection of 4-Hydroxy Tamoxifen has a limit of detec tion less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to i ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.5 ng/mL. in some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.4 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.2 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.1 ng/mL.

[8(528] In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy

Tamoxifen (Endoxifen) has a limit of detection less than or equal to 5 ng/mL. in some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of lS!-Desmeihyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4- Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen

(Endoxifen) has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of N-Desm.ethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.4 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.2 ng/mL. In some embodiments, the method of detection of N-Desmethyl- 4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.15 ng/mL.

[8029] In some embodiments, the method of detection of N-Desmethy 1-4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of defection of N-Desmethyl-4' -Hydroxy Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4'- Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of - Desmethyl-4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4' -Hydroxy Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4' -Hydroxy Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.5 ng/mL, In some embodiments, the method of detection of N-Desmethyl-4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.4 ng/mL.

[0030] In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 3 ng mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 1.2 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 0.5 ng/mL.

[0031] As used herein, the term "purification" or "purifying" does not refer to removing all materials from the sample other than the analyte(s) of interest. Instead, purification refers to a procedure that enriches the amount of one or more analyses of interest relative to one or more other components of the sample. Purification, as used herein, does not require the isolation of an analyte from all others. In preferred embodiments, a purification step or procedure can be used to remove one or more interfering substances, e.g., one or more substances that would interfere with the operation of the instruments used in the methods or substances that may interfere with the detection of an analyte ion by mass spectrometry.

[8032] As used herein, the term "about" in reference to quantitative measurements, not including the measurement of mass of an ion, refers to the indicated value plus or minus 10%. [8(533] As used herein, the term "substantially all" refers to any proportion greater than 50%, more preferably greater than 60%, more preferably greater than 70%, more preferably greater than 80%, and more preferably greater than 90%.

[8034] As used herein, the term "sample" refers to any sample that may contain the analyte of interest. As used herein, the term "body fluid or tissue" means any fluid or tissue that can be isolated from the body of an individual. For example, "body flu d or tissue" may include blood, plasma, serum, bile, saliva, urine, tears, perspiration, and the like. If solid tissue is to be analyzed, it may be processed to release a liquid fraction that could contain any analyte present in the tissue. The liquid fraction can then be subject to the methods described herein.

[8035] As used herein, the term "size separation technique" means any technique (physical or chemical) that allows for the separation of at least one species from a test sample based on any one or more of molecular weight and shape. Examples of such techniques include, but are not limited to, filtration, chromatography, and certain aspects of mass spectrometry.

[0036] As used herein, the term "chromatography" refers to a process in which a chemical mixture earned by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flo around, over, and/or through a stationary liquid or solid phase.

[8037] As used herein, the term "liquid chromatography" or "LC" means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). "Liquid chromatography" includes reverse phase liquid

chromatography (RPLC), high performance liquid chromatography (HPLC) and high turbulence liquid chromatography (HTLC).

[8038] As used herein, the term "high performance liquid chromatography" or "HPLC" refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column.

[8039] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique to identify compounds by their mass. MS refers to methods of filtering, detecting, and measuring ions based on their m/z. MS technology generally includes (1 ) ionizing the compounds to form charged species (e.g., ions); and (2) detecting the molecular weight of the ions and calculating their m/z. The compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. In general, one or more molecules of mterest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass ("m") and charge ("z"). See, e.g., U.S. Patent Nos. 6,204,500, entitled "Mass Spectrometry From Surfaces;" 6, 107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry;" 6,2.68,144, entitled "DNA Diagnostics Based On Mass Spectrometry;" 6, 124, 137, entitled "Surface-Enhanced

Photolabiie Attachment And Release For Desorption And Detection Of Analytes;" Wright et a!., Prostate Cancer and Prostatic Diseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis 21: 1 164-67 (2000).

[8048] As used herein, the term "operating in positive ion mode" refers to those mass spectrometry methods where positive ions are detected. Similarly, the term "operating in negative ion mode" refers to those mass spectrometry methods where negative ions are detected.

[8041] As used herein, the term "ionization" or "ionizing" refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Positive ions are those having a net positive charge of one or more electron units. Negative ions are those having a net negative charge of one or more electron units.

[8(542] As used herein, the term "electron ionization" or "EI" refers to methods in which an analyte of interest in a gaseous or vapor phase interacts with a flow of electrons. Impact of the electrons with the analyte produces analyte ions, which may then be subjected to a mass spectrometry technique.

[6043] As used herein, the term "chemical ionization" or "CI" refers to methods in which a reagent gas (e.g. ammonia) is subjected to electron impact, and analyte ions are formed by the interaction of reagent gas ions and analyte molecules.

[8044] As used herein, the term "fast atom bombardment" or "FAB" refers to methods in which a beam of high energy atoms (often Xe or Ar) impacts a non-volatile sample, desorbing and ionizing molecules contained in the sample. Test samples are dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyJ alcohol, 18-crown-6 crown ether, 2-nitropheiiyloctyl ether, sulfolane, diethanolamine, and triethanolamine. The choice of an appropriate matrix for a compound or sample is an empirical process. [8(545] As used herein, the term "matrix-assisted laser desorption ionization" or "MALDI" refers to methods in which a non- volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photo- ionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy-absorbing matrix, which facilitates desorption of analyte molecules.

[8046] As used herein, the term "surface enhanced laser desorption ionization" or "SELDl" refers to another method in which a non- volatile sample is exposed to laser irradiation, which desorbs and ionizes analytes in the sample by various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For SELDl, the sample is typically bound to a surface that preferent lly retains one or more analytes of interest. As in MALDI, this process may also employ an energy-absorbing material to facilitate ionization.

[8047] As used herein, the term "electrospray ionization" or "EST," refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber, which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

[8848] As used herein, the term "atmospheric pressure chemical ionization" or "APCI," refers to mass spectroscopy methods that are similar to ESI; however, APCI produces ions by ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electric discharge between the spray capillary and a counter electrode. Then ions are typically extracted into the mass analyzer by use of a set of differentially pumped skimmer stages. A counterflow of dry and preheated N₂ gas may be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than ESI for analyzing less-polar species.

[8049] The term "Atmospheric Pressure Photoionization" or "APPT" as used herein refers to the form of mass spectroscopy where the mechanism for the photoionization of molecule M is photon absorption and electron ejection to form the molecular M+. Because the photon energy typically is just above the ionization potential, the molecular ion is less susceptible to dissociation. In many cases it may be possible to analyze samples without the need for chromatography, thus saving significant time and expense. In the presence of water vapor or protic solvents, the molecular ion can extract H to form MH+. This tends to occur if M has a high proton affinity. This does not affect quantitation accuracy because the sum of M+ and MH+ is constant. Drag compounds in protic solvents are usually observed as MH+, whereas nonpolar compounds such as naphthalene or testosterone usually form M-K Robb, D.B., Covey, T,R. and Bruins, A.P. (2000): See, e.g., Robb et al, Atmospheric pressure photoionization: An ionization method for liquid chromatography- mass spectrometry. Anal. Chem. 72(15): 3653-3659.

[0050] As used herein, the term "inductively coupled plasma" or 'TCP" refers to methods in which a sample is interacted with a partially ionized gas at a sufficiently high temperature to atomize and ionize most elements

[0051] As used, herein, the term "field desorption" refers to methods in which a non-volatile test sample is placed on an ionization surface, and an intense electric field is used to generate anaiyte ions.

[0052] As used herein, the term "desorption" refers to the removal of an anaiyte from a surface and/or the entry of an anaiyte into a gaseous phase.

[0053] As used herein, the term "limit of quantification" or "LOQ" refers to the point where measurements become quantitatively meaningful. The anaiyte response at this LOQ is identifiable, discrete and reproducible with a precision of 20% and an accuracy of 80% to 120%.

[8054] In certain preferred embodiments of the methods disclosed herein, mass

spectrometry is performed in positi ve ion mode. In certain particularly preferred embodsments of the methods disclosed herein, mass spectrometry is performed using ESI.

[8055] In other preferred embodiments, a separately detectable internal standard is provided in the sample.

[8056] In one embodiment, the methods involve the combination of LC with mass spectrometry. In another preferred embodiment, the mass spectrometry is tandem mass spectrometry (MS/MS).

[0057] The summary of the invention described above is non-limiting and other features and advantages of the in v ention will be apparent from the following detailed description of the invention, and from the claims. DETAILED DESCRIPTION OF THE INVENTION

[ΘΘ58] Methods are described for quantitatively measuring tamoxifen and/or metabolites thereof in a patient sample. This quantitative measurement is achieved through the use of LC-MS/MS techniques. Prior to the use of LC-MS/MS, samples may be prepared by the following technique, or any portion thereof. A first purification of tamoxifen and/or metabolites thereof in a sample may be conducted through the use of a protein purification, filtration, or chromatography.

[0059] Any suitable size separation technique may be utilized, but in the examples that follow, both the first and second size separation techniques are filtration through a molecular weight cut-off filter. It is also possible, as discussed in the Examples that follow, to select a molecular weight cut-off filter with an appropriate molecular weight cut-off such that the same filter can be used for both the first size separation and the second size separation.

[0068] LC, most preferably liPLC, is utilized, may be utilized either alone or in combination with other purification methods, to purify selected analytes. This purification is combined with MS/MS, thereby providing an assay system for quantifying selected analytes in a test sample. The quantity of the selected analytes in the sample is then used to determine the quantify of tamoxifen in the original test sample. The quantitation methods provided herein have enhanced specificity and are less subject to methodological problems (such as antibody interference).

[0061] Suitable samples may include any test sample that may contain the analyte of interest, in some preferred embodiments, a sample is a biological sample; that is, a sample obtained from any biological source, such as an animal, a cell culture, an organ culture, and the like. In certain preferred embodiments, samples are obtained from a mammalian animal, such as a dog, cat, horse, etc. Particularly preferred mammalian animals are primates, most preferably humans. Particularly preferred samples include blood, plasma, serum, urine, saliva, tears, cerebrospinal fluid, or other body fluid or tissue samples. Such samples may be obtained, for example, from a patient; that is, a living person presenting oneself in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition. The test sample is preferably obtained from a patient, for example, serum or plasma.

Sample Preparation for Mass Spectrometry [8(562] Samples may be processed or purified to obtain preparations that are suitable for analysis by mass spectrometry. Such purification will usually include chromatography, such as liquid chromatography, and may also often involve an additional purification procedure that is performed prior to chromatography. Various procedures may be used for this purpose depending on the type of sample or the type of chromatography. Examples include filtration, centrifugation, combinations thereof and the like.

[8(563] Filtration is one preferred method of preparing a test sample, especially a biological test sample, such as serum or plasma, for chromatography . Such filtration is carried out by filtering a test sample through a molecular weight cut-off filter to separate species with molecular weights higher than the filter's cut-off from those with molecular weights lower than the filter's cut-off. The test sample remaining above the filter following complete (or near complete) filtration is substantially free of potentially interfering species with molecular weights lower than the filter's cut-off.

[8864] Various methods have been described involving the use of HPLC for sample cleanup prior to mass spectrometry analysis. See, e.g., Taylor et al., Therapeutic Drug Monitoring 22:608-12 (2000) (manual precipitation of blood samples, followed by manual C18 solid phase extraction, injection into an HPLC for chromatography on a CI 8 analytical column, and MS/MS analysis); and Saim et al, Clin. Therapeutics 22 Supl. B:B7f-B85 (2000) (manual precipitation of blood samples, followed by manual C18 solid phase extraction, injection into an HPLC for chromatography on a CI 8 analytical column, and MS/MS analysis). One of skill in the art may select HPLC instruments and columns that are suitable for use in the methods. The chromatographic column typically includes a medium (i.e., a packing material) to facilitate separation of chemical moieties (i.e., fractionation). The medium may include minute particles. The particles include a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties. One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded surface. Alkyl bonded surfaces may include C-4, C-8, or C-18 bonded alkyl groups, preferably C-8 bonded groups. The chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an effluent that includes the fractionated sample.

[8065] In certain embodiments, an analyte may be purified by applying a sample to a column under conditions where the analyte of interest is reversibly retained by the column packing material, while one or more other materials are not retained. In these embodiments, a first mobile phase condition can be employed where the analyte of interest is retained by the column and a second mobile phase condition can subsequently be employed to remove retained material from the column, once the non-retained materials are washed through. Alternatively, an analyte may be purified by applying a sample to a column under mobile phase conditions where the analyte of interest elutes at a differential rate in comparison to one or more other materials. Such procedures may enrich the amount of one or more analyses of interest relative to one or more other components of the sample.

[8(566] In one embodiment, the sample to be analyzed is applied to the column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port. Different solvent modes may be selected for eluting the analytes of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode. In preferred embodiments, HPLC is performed on an analytical HPLC system with a C8 solid phase using 0.2% formic acid in HPLC Grade Ultra Pure Water and 0.2% formic acid in 100% methanol as the mobile phases.

[8(567] Numerous column packings are available for chromatographic separation of samples and selection of an appropriate separation protocol is an empirical process that depends on the sample characteristics, analyte of interest, presence of interfering substances and their characteristics, etc. Commercially available HPLC columns include, but are not limited to, polar, ion exchange (both cation and anion), hydrophobic interaction, phenyl, C-2, C-8, C-18, and polar coating on porous polymer columns.

[8868] In one embodiment, the HPLC column has a C8 solid phase with a median particle size of 5μηι (nominal) and a median particle pore size of 100 A. In a preferred embodiment the column dimensions are 1.0 mm ID x 50 mm length (Pbenomenex Corp. Luna 5μ C8(2) 100 A New Column 50 x 1.0 mm, Pbenomenex Cat. No. 00B-4249-A0 or equivalent).

[8069] During chromatography, the separation of materials is effected by variables such as choice of eluent (also known as a "mobile phase"), choice of gradient elution and the gradient conditions, temperature, etc.

Detection and Quantitation by Mass Spectrometry

[8078] In various embodiments, analytes may be ionized by any method known to the skilled artisan. Mass spectrometry is performed using a mass spectrometer, which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis. Ionization sources used in various MS techniques include, but are not limited to, electron ionization, chemical ionization, eiectrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCT), photoionization, atmospheric pressure photoiomzaiion (APPI), fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption ionization (MALDl), field ionization, field desorption, thermospray/plasmaspray ionization, surface enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP) and particle beam ionization. The skilled artisan will understand ihat the choice of ionization method may be determined based on the analyte io be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc.

[0071] In preferred embodiments, analytes are ionized by electrospray ionization (ESI) creating analyte precursor ions. In related preferred embodiments, analyte precursor ions are in a gaseous state and the inert collision gas is argon.

[0072] After the sample has been ionized, the positively charged ions thereby created maybe analyzed to determine m z. Suitable analyzers for determining m/z include quadripole analyzers, ion trap analyzers, and time-of- flight analyzers. The ions may be detected using one of several detection modes. For example, only selected ions may be detected using a selective ion monitoring mode (SIM), or alternatively, multiple ions may be detected using a scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). In preferred embodiments, ions are detected using SRJVL

[8073] Preferably, m/z is determined using a quadrupole instrument. In a "quadmpole" or "quadmpole ion trap" instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the R.F signal, and m/z. The voltage and amplitude may be selected so that only ions having a particular m/z travel the length of the quadrupole, while all other ions are deflected. Thus, quadmpole instruments may act as boih a "mass filter" and as a "mass detector" for ihe ions injected into the instrument.

[8074] One may enhance the resolution of the MS technique by employing "tandem mass spectrometry," or "MS/MS." In this technique, a precursor ion (also called a parent ion) generated from a molecule of interest can be filtered in an MS instrument, and the precursor ion subsequently fragmented to yield one or more fragment ions (also called daughter ions or product ions) that are then analyzed in a second MS procedure. By careful selection of precursor ions, only ions produced by certain analytes are passed to the fragmentation chamber, where collision with atoms of an inert gas produce the fragment ions. Because both the precursor and fragment ions are produced in a reproducible fashion under a given set of ionization/fragmentation conditions, the MS/MS technique may provide an extremely powerful analytical tool. For example, the combination of filtration/fragmentation may be used to eliminate interfering substances, and may be particularly useful in complex samples, such as biological samples.

[8075] Additionally, recent advances in technology, such as matrix-assisted laser desorption ionization coupled with time-of-flight analyzers ("MALDI-TOF") permit the analysis of analytes at femtomole levels in very short ion pulses. Mass spectrometers that combine time- of-flight analyzers with tandem MS are also well known to the artisan. Additionally, multiple mass spectrometry steps may be combined in methods known as "MS/MS". Various other combinations may be employed, such as MS/MS/TOF, MALDI/MS/MS/TOF, or

SELDI/MS/MS/TOF mass spectrometry.

[8076] The mass spectrometer typically provides the user with an ion scan; that is, the relative abundance of each ion with a particular m/z over a given range (e.g., 400 to 1600 amu). The results of an analyte assay, that is, a mass spectrum, may be related to the amount of the analyte in the original sample by numerous methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion may be compared to a table that converts that relative abundance to an absolute amount of the original molecule. Alternatively, molecular standards may be run with the samples and a standard curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ion may be converted into an absolute amount of the original molecule. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating the quantity of tamoxifen.

Methods of generating and using such standard curves are well known in the art and one of ordinary skill is capable of selecting an appropriate internal standard. Numerous other methods for relating the amount of an ion to the amount of the original molecule will be well known to those of ordinary skill in the art.

[8077] One or more steps of the methods ma be performed using automated machines. In certain embodiments, one or more purification steps are performed on-line, and more preferably all of the LC purification and mass spectrometry steps may be performed in an online fashion. [8(578] In certain embodiments, techniques such as MS/MS are used to isolate precursor ions for further fragmentation. In these embodiments, collision activation dissociation (CAD) may be used to generate the fragment ions for further detection. In CAD, precursor ions gain energy through collisions with an inert gas, and subsequently fragment by a process referred to as "unimolecular decomposition". Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to increased vibrational energy. In alternative embodiments, electron transfer dissociation (ETD) may be used to generate the fragment ions. In ETD, radical anions are used to transfer electrons to multiply charged peptide or protein cations resulting in random cleavage along the peptide backbone.

[8079] In particularly preferred embodiments, analyte is detected and/or quantified using LC-MS/MS as follows. An analyte enriched sample prepared as described above is subjected to LC. The flo of liquid solvent from the chromatographic column enters the heated nebulizer interface of a LC-MS/MS analyzer and the solvent analyte mixture is converted to vapor in the heated tubing of the interface. The analyte contained in the nebulized solvent, is ionized by the corona discharge needle of the interface, which applies a large voltage to the nebulized solvent/analyte mixture. The ions pass through the orifice of the instrument and enter the first quadrupofe. Quadrupoles 1 and 3 (QI and Q3) are mass filters, allowing selection of ions (i.e., "precursor" and "fragment" ions) based on their m/z. Quadrupole 2 (Q2) is the collision cell, where ions are fragmented. Q 1 selects for ions with m/z of precursor ions. Selected precursor ions are allowed to pass into the collision chamber (Q2), while ions with any other m/z collide with the sides of Ql and are eliminated. Precursor ions entering Q2 may be fragmented with collision activated dissociation (CAD) through collisions with neutral argon gas molecules. Alternatively, if the precursor ions entering Q2 are multiply charged cations, they may be fragmented with electron transfer dissociation (ETD). The fragment ions generated are passed into Q3, where selected fragment ions are collected while other ions are eliminated.

[808ft] Using standard methods well known in the art, one of ordinary skill is capable of identifying one or more fragment ions of a particular precursor ion that may be used for selection in Q3. A specific iragment ion is one that will not be formed in significant amounts by other molecules with similar molecular structures. In contrast, a non-specific fragment ion is one that is formed by molecules other than the desired analyte. Suitable specific fragment ions can be identified by testing various molecular standards to determine whether fragment ions formed by a selected analyte are also formed by other molecules with similar structures or features. Preferably, at least one fragment ion specific for ions with m/z corresponding to that of analyte ions are identified.

[8081] As ions collide with the detector they produce a pulse of electrons that are converted to a digital signal. The acquired data is relayed to a computer, which plots ion counts per unit time. The areas under the peaks corresponding to particular ions, or the amplitude of such peaks, are measured and the area or amplitude is correlated to the amount of the analyte of interest. In certain embodiments, the area under the curves, or amplitude of the peaks, for fragment ion(s) and/or precursor ions are measured to determine the amount of analytes with m/z. As described above, the relative abundance of a given ion may be converted into an absolute amount of the original analyte using calibration standard curves based on peaks of one or more ions of an internal molecular standard. The absolute amount of an analyte detected by LC-MS/MS can then be converted into an absolute amount of analyte that was present in the original test sample.

[8(582] In some embodiments, the method provided herein has sensitivity measured by limit of quantitation (LOQ). In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 5 ng/mL, In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL.

[8083] In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl Tamoxifen has a limit of quantitation less than or equal to 1 .5 ng mL. [8(584] In some embodiments, the method of quantitation of 4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of 4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of 4' -Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4 '-Hydroxy Tamoxifen has a limit of quantiiation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of 4'-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of 4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantiiation of 4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of 4 '-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.4 ng/mL. In some embodiments, the method of quantitation of 4'-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.2 ng/mL.

[8085] In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.4 ng mL. In some embodiments, the method of quantitation of 4-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.2 ng/mL. [8(586] In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal io 5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen

(Endoxifen) has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantiiation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4- Hydroxy Tamoxifen (Endoxifen) has a limit of quantiiation less than or equal to 2 ng mL. In some embodiments, the method of quantitation of N-Desmethy 1-4 -Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 1.5 ng/mL. In some

embodiments, the method of quantitation of N-DesmethyI-4-Hydroxy Tamoxifen

(Endoxifen) has a limit of quantitation less than or equal io 1 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 0.5 ng mL. In some embodiments, the method of quantitation of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of quantitation less than or equal to 0.4 ng/mL.

[8087] In some embodiments, the method of quantitation of N-Desmethyl-4'-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4' -Hydroxy Tamoxifen has a limit of quantitation less than or equal to 4 ng/mL. In some embodiments, the method of quantitation of N- Desmethyl-4' -Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4' -Hydroxy Tamoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4'-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 2. ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4' - Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4^'' -Hydroxy Tamoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4'-Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.5 ng/mL. In some embodiments, the method of quantitation of N-Desmethyl-4' - Hydroxy Tamoxifen has a limit of quantitation less than or equal to 0.4 ng/mL. [8(588] In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 5 ng/niL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation Jess than or equal to 4 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 3 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 2 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 1.5 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 1.2 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 1 ng/mL. In some embodiments, the method of quantitation of norendoxifen has a limit of quantitation less than or equal to 0.5 ng/mL.

[8089] In some embodiments, the method provided herein has sensitivity measured by limit of detection (LOD). In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 1 ng mL. in some embodiments, the method of detection of tamoxifen has a limit of detection less than or equal to 0.6 ng/mL.

[8098] In some embodiments, the method of detection of N-Desmethyl T amoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N- Desmethyl Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of N- Desmethyl Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of def ection of N-Desmethyl Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of - Desmethy] Tamoxifen has a limit of detection less than or equal to 0.6 ng/mL.

[8091] In some embodiments, the method of detection of 4" -Hydroxy Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of 4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of 4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4' -Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of 4' -Hydroxy Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of 4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to I ng/mL. In some embodiments, the method of detection of 4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of 4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.4 ng/mL. In some embodiments, the method of detection of 4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.2 ng/mL. In some embodiments, the method of detection of 4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.1 ng/mL.

[8(592] In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 2 ng mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.4 ng/mL. In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0,2 ng/mL, In some embodiments, the method of detection of 4-Hydroxy Tamoxifen has a limit of detection less than or equal to 0, 1 ng/mL,

[8093] In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy

Tamoxifen (Endoxifen) has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of N-Desmethy3-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 3 ng/mL. I some embodiments, the method of detection of N-Desmethyi-4- Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 3 ng/mL, In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen

(Endoxifen) has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 1 ng/mL. in some embodiments, the method of detection of N-Desmethy]-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.5 ng mL. In some embodiments, the method of detection of N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.4 ng/mL. In some embodiments, the method of detection ofN-Desmethyi-4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.2 ng mL. In some embodiments, the method of detection of N-Desmethyl- 4-Hydroxy Tamoxifen (Endoxifen) has a limit of detection less than or equal to 0.15 ng/mL.

[8094] In some embodiments, the method of detection of N-Desmethyl-4' -Hydroxy Tamoxifen has a limit of detection less than or equal to 5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4'- Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection ofN- Desmethy] -4 '-Hydroxy Tamoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4' -Hydroxy Tamoxifen has a limit of detection less than or equal to 1.5 ng/mL. In some embodiments, the method of detection of r>l-Desmethyj-4'-Hydroxy T amoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection ofN-Desmethyl-4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.5 ng/mL. In some embodiments, the method of detection of N-Desmethyl-4'-Hydroxy Tamoxifen has a limit of detection less than or equal to 0.4 ng/mL.

[8095] In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 5 ng mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 4 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 3 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 2 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 1.5 ng mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 1.2 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 1 ng/mL. In some embodiments, the method of detection of norendoxifen has a limit of detection less than or equal to 0.5 ng/mL.

[8096] The following examples serve to illustrate the invention. These examples are in no way intended to limit the scope of the methods.

EXAMPLES

Example 1 : Determination of tamoxifen and its metabolites

[8097] In the following procedure, tamoxifen and its metabolites are extracted from serum. The semm is added to a filter plate, and then mixed with acetonitrile/IS, which then forms a precipitate. The mixture is then placed on a positive pressure manifold, and the organic fraction is passed through to a collection plate. The plate is lidded, and then placed on the Cohesive system for injection onto MS/MS. The MS is in APCT-positive mode. The quantitation is based upon unique parent- roduct transitions. Those analytes with similar transitions were separated chromatographically.

[8(598] Patient Pre aration Component Special Notations

Fasting/Special Diets N/A

Specimen Collection N/A

and/or Timing

Special Collection N/A

Procedures

Other No aromatase inhibitors.

Specimen T e & Handling

agent Summary

Supplier & Catalog Number Quantity Ammonium Formate Sigma, 17843-250G or veriiied 250 Grams equivalent

Formic Acid Sigma, F0507- 100mL or l OO mL

verified equivalent

Ethanol, Absolute, Pharmeo-AAPER, 1 pint Anhydrous, 200 Proof Cai#l 1 1000200 or verified

equivalent

Acetonitrile, HPLC B&J, Cat #015-4 or verified

4 Liters Grade equivalent

Biocell Serum, Hitman

Biocell Labs, Cat # 1 121 -00 or

Serum, Stripped and 4 Liter verified equivalent

Delipidized (Opticlear)

Toronto Research Labs (TRC),

Tamoxifen Cat#T006000 or verified 25 Grams equivalent

Toronto Research Labs (TRC),

N-Desmethyl Tamoxifen

Cat#D293900 or verified 50 mg HQ

equivalent

Toronto Research Labs (TRC),

-Desmethyj-4-Hydroxy

Cat#D292043 or verified 50 mg Tamoxifen

equivalent

Toronto Research Labs (TRC),

N-Desrnetbyi-4 '- Cat#D292041 or verified 1 0 mg Hydroxy Tamoxifen

equivalent

Toronto Research Labs (TRC),

(Z)-4-Hydroxy

Cat#H954725 or verified 100 mg

Tamoxifen

equivalent

Toronto Research Labs (TRC),

4 ' -Hydroxy Tamoxifen Cat#H954730 or verified 25 mg equivalent

Toronto Research Labs (TRC),

(E/Z)-Tamoxifen-d5 Cat#T006007 or verified 10 mg equivalent Toronto Research Labs (TRC),

N-Desmethyl

Cat#D293902 or verified 1 0 nig Tamoxifen-d5

equivalent

Toronto Research Labs (TRC),

-Desmethyl-4-Hydroxy

Cat#D292044 or verified 1 0 mg Tamoxifen-d5

equivalent

Toronto Research Labs (TRC),

N-Desmethyl-4'- Cat#D291867 or verified 1 0 mg Hydroxy Tamoxifen-d3

equivalent

Toronto Research Labs (TRC),

(Z)-4-Hydroxy

Cat#H954757 or verified 25 mg Tamoxifen-d5

equivalent

Toronto Research Labs (TRC),

4 ' -Hydroxy Tamoxifen- Cat#H954757 or verified 0 mg cl6

equivalent

[00101] Calibrators/Standards Used

[00102] A 12-point calibration is used for each analyte. Initially, only one standard is made (Std- 12), with a series of dilutions performed to generate the remaining standards. The standard (#12) is to be removed from the -70°C freezer and thawed. While thawing, label twelve 12x75 mm tubes.

[00103] Add 3.0 ml. of std- 12 to tube 12. From this standard, you will follow the table below^r to create the standard curve. A standard curve is to be generated with each assay. Place the initial standard back in the -60 to -90°C freezer.

'-Hydroxy Tamoxifen 200 40 uL 80104] Once these analytes have been added, QS to 200 mL with Biocell serum. Mix, then aliquot into 15 mL centrifuge tubes. Label (Std- 12), then place the tubes in the -60°C to -90°Cfreezer for storage. Stable for 1 year.

[00105] Standard Target Concentrations concentrations in

ng/mL.

CaJibration_^Curye Diiutiorts:

libration Procedure

Refer to the dilution table in section 5.2. Freeze/Thaw studies were conducted, and showed that the analytes were stable over 5 cycles. Always return Std- 12 to the - 60°C to -90°C freezer as soon as you are finished with it. - Use Quadratic curve fit for all analytes, except Endoxifen (linear). Origin's are set to ignore, and weighting is set to 1/y.

[00107; EQUIPMENT and SUPPLIES

[80188; Assay Platform

0010 ; A Thermo LC/MS/MS system containing the following modules was used for this assav:

Thermo Quantum Ultra (Serial #022-TQU01376)

Cohesive Aria TLX-4 (Serial # SJCTX457)

LC Quant Software (Xcalibur, Thermo Fisher)

Aria 1.6 Software

Agilent GT312 Binary Pumps

Thermo APCT Source

[00110] Equipment

[80111] Supplies

SuDDlv Supplier Cat #

250 uL Rainen Pipette Tips Rainen HR-250

1000 uL Pipette Tips Rainen HR-1000

Thermo 96 Deep Well Plates Thermo Scientific P/N 260252

Thermo Pre-Slit Well Cap for 96 Well PP Thermo Scientific P/N 27601 1

Plate

Sirocco Protein Precipitation Plate, 5/pk Waters Corp P/N 186002448 [8(5112] Mass Spectrometry

[00114] EXPECTED VALUES

[00115] Reference Ranges:

Tamoxifen: 12.54-233.07 ng/ml.

N-Desmethyl Tamoxifen: 2.59-373.96 ng/mL

4'-Hydroxy Tamoxifen: 0.4-6.33 ng/mL

4-Hydroxy Tamoxifen: 0.24-5.05 g/mL

N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen): 0.93-43.19 ng/mL N-Desmethy3-4'-Hydroxy Tamoxifen: 1.17- 19.95 ng/mL

[00116] Analytical Measurement Range (AMR)

Tamoxifen: 1.47- 1500 ng/mL

N-Desmethyl Tamoxifen: 1.47- 1500 ng/mL

4'-Hydroxy Tamoxifen: 0.2-200 ng/mL

4-Hydroxy Tamoxifen: 0.2-200 ng/mL

N-Desmethyl-4-Hydroxy Tamoxifen (Endoxifen): 0.39-400 N-Des methyl -4 '-Hydroxy Tamoxifen: 0.39-400 ng/mL [00117] Precision: Inter- (span of assay) and intra-assay ( I day) precision studies were performed using (he low, medium, and high controls. All analytes showed a <12% CV over the span of the validation,

[80118] Interfering Substances: Mild or moderate icteric and lipemic samples are acceptable. Hemolytic samples are not acceptable, as they will clog the filter during sample prep.

Grossly hemoiyzed, icteric, and lipemic samples are not acceptable.

[00119] Clinical Sensitivity (LOQ):

Tamoxifen: 1.47 ng/mL

N-Desmethyl Tamoxifen: 1.46 ng/niL

4 '-Hydroxy Tamoxifen: 0.2 ng/mL

4-Hydroxy Tamoxifen: 0.2 ng/mL

N-Desmethy3-4-Hydroxy Tamoxifen (Endoxifen): 0.39 ng/mL

N-Desmethyl-4'-Hydroxy Tamoxifen: 0.39 ng/mL

Example 2: Validation of tamoxifen and its metabolites assay

[00120] This report contains a detailed summary of the validation for Tamoxifen and its 5 main phase I metabolites by LC/MS/MS. The assay is a laboratory-developed test.

[80121] Methodology: Tamoxifen and its five main phase I metabolites (N- desmethyltamoxifen, N-Desmethyl-4-hydroxytamoxifen, N-desmehtyl-4'- hydroxytamoxifen, 4-hydroxytamoxifen, and 4'-hydroxytamoxifen) are extracted from serum. The extraction is a protein precipitation, followed by filtration. Analysis and quantitation is then performed by LC/MS/MS.

[80122 Kit or Reagents

Toronto Research Labs (T C),

Tamoxifen

Cat#T006000

Toronto Research Labs (TRC),

N-Desmeihyi Tamoxifen HQ

Cat#D293900

Toronto Research Labs (TRC),

N-Desmethyl-4-Hydroxy Tamoxifen

Cat#D2.92043

Toronto Research Labs (TRC),

N-Desmethy 1-4 ' -Hydroxy Tamoxifen

Cat#D292041

Toronto Research Labs (TRC),

(Z)-4-Hydroxy Tamoxifen

Cat#H954725

Toronto Research Labs (TRC),

4 '-Hydroxy Tamoxifen

Cat#H954730

Toronto Research Labs (TRC),

(E/Z)-Tamoxifen-d5

Cat#T006007

Toronto Research Labs (TRC),

N-Desmethyl Tamoxifen-d5

Cat#D293902

Toronto Research Labs (TRC),

N-Desmethyl-4-Hydroxy Tatnoxifen-d5

Cat#D292044

Toronto Research Labs (TRC),

N-Desmethyl-4' -Hydroxy Tamoxifen-d3

Cat#D291867

Toronto Research Labs (TRC),

(Z)-4-Hydroxy Tamoxifen-d5

Cat#H954757

Toronto Research Labs (TRC),

4 ' -Hydroxy Tamoxi fen-d6

Cat#H954757

[8(5123] Precision Study for Laboratory Developed Tests (LDT)

[00124] Within Run PrecisioniLow, medium, and high controls were analyzed (10) within one run. All QC fell within acceptability criteria (<20% CV).

4-OH" N-DM-4'OH" 4ΌΗ- -DKS-

Endoxiferi Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen

Low Control (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL)

1 11.479 1.582 10.170 1.926 34,728 32.814

2 7.510 1.806 8.915 1 757 32.952 39.576

3 8.580 1.751 9.064 .552 36.389 37.948 4 9.156 1.833 9.196 1.641 35.488 33.814

5 9.805 1.844 9.221 1.643 33.879 31.212

6 9.088 2.132 8.785 1.758 32,435 34.300

7 8.821 2.079 8.569 .559 33.555 36.784

8 7.304 2.383 8.440 ■ .994 35.117 33.327

9 9.921 2.1 19 9.136 1.745 37.190 33.794

10 8.550 2.192 8.102 1.845 38.720 36.350

Mean 9.02 1.97 8.98 1.74 35.05 34.99

Stdev 1.21 0.24 0.56 0.15 1.97 2.58

% 13.41 12.42 6.25 8.51 a 63 7.37

4-OH- -DM-4'OH- 4ΌΗ- Η~ΏΜ-

Endoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen

High Contra! (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL)

I 189.670 93.480 188.415 92.408 658.605 858.395

2 20 .279 91.872 89.325 91.608 755.094 882.797

3 189 009 95 «69 174.819 84,116 724,537 780 958

4 20 .229 92.422 90.193 87.989 719.015 887.024

5 1 8.358 88.508 ■81.242 92.937 683.269 763.216

6 1 0.784 85.174 177.770 85.490 721.1 1 823.250

7 184.641 98.301 72.371 86.206 706.818 783.975

8 205 803 89.703 184.633 84,096 7 2,341 784 042

9 190.205 94.532 164.908 84.977 723.057 805.741

10 190.731 89.605 189.710 96.315 737.294 819.099 ft/!ean 192.17 91.96 181.34 88.61 714.12 818.85

Stdev 10.14 3.86 8.68 4,36 27,01 44.03 % CV 5.28 4.20 4.79 4.92 3.78 5.38

[00125] Total Precisio Total precisior! was based on all QC run on all assays during the validation process. Acceptability was based on < 20% CV. All QC fell within this criteria.

4-QH- -DM-4OH- 4ΌΗ- Η-ΏΜ-

Low Endoxifert Tamoxifen Tamoxifen Tamoxifert Tamoxifen Tamoxifen

Contra! (ng/miL) (ng/mL) (ng/mL) (rtg/mL) (ng/iTiL) (ng/mL)

1 1 .592 2.285 8,663 1 .525 36.145 35.525

2 1 1 .138 2.277 9.762 2.168 40.870 38. 52

3 8.890 2.519 ■ 0.094 2.145 40.451 37.248

^ 10.022 2.41 0.096 2.363 34.538 38.348

5 10.377 1 .964 9.531 2,130 40.335 37.198

1 8.917 2. 69 8.172 ,932 34.775 35.754

2 9.833 1 .926 9.707 .953 37.485 34.955

3 8.974 2.066 9.451 2.239 36.460 36. 76

4 9.810 2.035 8.760 1 .621 32.040 32.630

5 10.367 1 .751 8,983 1 .847 36.1 14 37.263

1 12 282 2.410 8,462 2.106 34.382 33.980

2 8.801 2.418 9.214 1 .696 38.913 37.524

3 10.512 1 .650 9.246 2.038 35.886 39.920

^ 9.479 2.072 8,300 1 .925 40.630 40.013

5 9.487 2.094 9.062 2,260 35.468 40.544

1 13.192 2.172 9.722 2,067 34.702 36.737

2 1■ .281 1 .876 9.876 1 .723 31 .707 36.750

3 9.727 1 .778 9.547 2.085 38.602 34.458

4 10.920 1 .956 9.623 1 ,902 35.276 36.427

5 10 765 1 .878 10.720 2.196 32.678 37.328

1 9.905 2.267 9.573 1 .756 34.575 40.865

2 9.517 2.419 10.397 1 .852 38.703 38.931

3 10.592 2.089 10.440 1 .768 36. 04 39.576

4 9.705 2.161 0,924 1 .881 37.768 40.039

5 Q ?gg 1 .964 9.492 .660 35.499 37.825

1 10.312 2.195 .309 1 .952 37.109 37.409

2 8.540 2.084 10.329 1 .848 32.896 39.378

1 9.971 2.699 10.21 1 ,968 37. 76 38.498

2 10 845 2.264 8.237 1 .985 38.033 42.613

3 9.275 2.184 8.027 1 .590 32.633 34.847

4 7.925 2.569 7.925 2.166 35.327 35.302

5 8.041 2.312 7.885 1 .891 31 .681 36.267

1 8.636 2.075 9,928 2.023 34.570 38.068

2 10.653 2.122 10.316 2.038 39.078 38.219

1 10.003 2.559 9.009 2.401 41 .780 38.067

2 13.146 2.143 9.527 2.285 37.945 36.085 1 9.319 2.202 9.477 2.347 34.554 38.110

2 8.029 1.836 8.287 1.789 33.201 30.038

3 9.012 2.140 8,720 1.974 35.778 34.251

4 8.299 2.037 9.456 2.119 38.542 37.302

1 9.Θ06 2.449 10.127 2.619 38.680 42.268

2 11.108 2.410 9.1 18 2.570 38.456 42.861

3 .502 2.288 8,941 2.224 36.295 39.003

4 9.983 2.536 9.583 2,301 39.444 42.359

1 10.863 2.027 9.7 3 1 .955 39.137 36.636

2 9.912 2.046 8.556 2.133 35.665 38.773

·ι 9.603 2.247 7.986 2.063 42.388 39.865

2 9.305 2.071 9,373 2.197 37.447 39.204

1 8 518 2.203 9.189 2.097 36.600 34.954

2 8.727 2.108 8.134 1.868 33.344 33.870

3 9.676 2.242 8.135 2.234 34.319 35.267

4 8.867 2.041 7,973 2.008 36.328 36.998

1 8 988 2.006 10.200 2.013 37.349 36.239

2 9.274 1.870 9.697 1.967 38.264 38.313

1 9.503 1.919 9.453 2.068 43.706 33.935

2 9.703 2.007 8.722 1.874 40.296 34.916

Mean 9.87 2.15 9.31 2,03 36.75 37.39

Std Dev 1.15 0.22 0,83 0.23 2.77 2.55

CV 11.85 10.3S 8.96 11.29 7.53 e.82

4-OH- N-DM-4OH- 4ΌΗ-

Middle Endoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen

Controi (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/m L) (ng/mL)

1 87.010 39.746 89.637 42.407 202.496 210.097

2 94.595 39.218 101.461 43.252 220.163 203.365

3 96.338 41.161 03.091 41.130 210.035 237.872

4 100.952 40.926 97.656 45.079 94.037 203.374

5 92.546 42.034 90.246 41.105 180.896 234.350

I 86.522 38.422 82.806 37.133 1 0.911 189.278

2 86.982 35.883 89,7 9 35.175 182.954 200.267

3 94.330 37.402 83.686 38. 87 87.342 186.339

4 86.088 37.256 90.762 37.099 177.586 67.783

5 90.805 36.694 88.020 37.999 186.762 1 6.707

1 88.741 38.437 77,735 34.791 188.429 170.437

2 93.659 38.068 86.365 37.361 182.615 185.327

3 89.770 39.654 76.566 33.949 184.621 210.695

94.247 38.336 80.834 35.738 82.464 201.245

5 88.359 39.617 79.947 38.285 196.110 185.179 1 96.0 3 38.875 94.650 38.235 192.418 1 8.464

2 89.914 35.280 74.974 34.501 195.061 174.347

3 90.408 36.250 94,097 36.874 192.154 180.649

4 86.933 35.653 88.543 37.264 198.757 172.231

5 105.603 37.055 96.923 39.572 166.573 188.035

·ι 96.715 38.736 91.402 38.503 193.668 192.852

2 85.550 38.719 91 ,593 38.211 192.601 203.520

3 96.364 40.895 97.744 40.532 203.027 210.815

4 89.677 39.397 77,862 36.283 0.794 91.809

5 84.419 36.150 87.890 36.258 203.371 192.906

1 87.929 38.550 88.816 36.835 181.353 197.693

2 74.719 36.294 87.041 34.933 179.365 190.056

1 83.567 36.801 83.282 39.022 77.547 77.361

2 75.551 33.187 85.481 34.572 153.055 159.856

3 87.804 38.901 71.354 28.960 166.592 192.261

4 82.223 37.510 72.808 29.301 166.157 179.546

5 85.341 39.784 71 ,964 30.776 171.216 205.388

1 88.004 33.537 90.680 33, 57 5.955 87,743

2 86.158 39.113 89.815 35.488 184.322 189. 94

1 88.728 41.774 76.301 35.150 179.239 21 .475

2 100.988 37.906 80.538 36.629 197.328 202.671

1 84.292 35.389 90.935 35.702 84.883 189.455

2 88.520 34.514 95.314 35.561 183.190 172.592

3 83.439 37.285 95.351 37.955 180.218 1 3.995

4 86.701 37.091 88. 60 36.253 192.596 180.127

1 86.049 37.984 83.921 36.871 72.997 224. 2

2 91.295 39.390 92.291 39.164 185.491 202.409

3 88.654 38.989 85. 21 38.139 177.713 213.566

4 89.973 40.718 93.456 39.341 197.407 215.637

1 93.239 42.549 91.506 38.826 181.470 205.651

2 98.907 38.658 82,931 36.807 8.912 216.152

1 93.912 38.063 89.926 35.099 188.095 222.914

9 83.242 34.984 88.570 37.402 200.103 210.098

1 96.655 38.764 81 ,895 36.921 173.766 179.647

2 93.507 36. 78 87.743 37.688 169.649 6.749

3 97.061 39.966 86.633 39.299 174.694 195.119

97.7Θ0 41.871 92.541 40.805 198.256 192.921

1 88.496 38.325 87.354 37.319 189.199 184.837

2 85.028 38.332 87.345 38.125 177.596 193.024

1 86.758 38.894 88.755 37.777 98.350 7.001

2 87.200 36.649 86.536 37.594 205.277 185.319

IWeara 89.90 38.18 87.30 37.19 86.75 194.62

Std Dev 5.88 2.08 7.08 2.91 12.48 16.69

CV S.54 5.44 8.08 7.82 6.68 3.58 4-OH- N-D -4OH- 4ΌΗ-

High Eri oxifers Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen

Corttro! (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL)

1 207.479 101 732 95.254 102.024 799.588 735 «69

190.387 102.016 226.236 108.850 860.365 798.839

3 202.213 98.424 199.550 101.300 856.971 783.327

4 210.429 102.722 202.273 106.471 811.375 829.7 1

5 212.515 109.031 222.061 111.194 857.859 859.031

1 196 747 103.01 204.676 99.578 816.049 747.992

2 206.775 95.510 180.867 87.664 710.089 875.109

3 186.389 95.141 195.533 88.284 671.914 798.004

4 184.079 93.074 170.879 85.515 680.514 748.155

5 196.620 93.918 166.410 9 .662 696.733 813.829

1 181.815 101.602 205.865 92.189 786.902 889.986

204.310 94.568 194.635 92.144 823.327 787.045

3 203 649 95.490 182.781 85.872 790.504 798.055

4 205.176 92.576 189.464 89.338 758.539 768.107

5 202.342 96.793 76.749 92.032 846.377 727.994

1 198.640 98.625 196.187 95.798 810.498 728.474

2 198.899 91.019 87.599 100. 36 751.812 805.304

3 199.035 84.927 179.312 95.215 737.664 770.813

4 212.276 84.154 190.044 100.630 734. 51 886.035

5 200 109 92.086 4.237 99.469 760.246 872.936

1 200.469 89.448 199.269 91.661 729.412 834.619

2 202.977 98.208 199.034 96.375 790.310 799.638

3 205.475 93.973 192.105 92.720 798.244 810.004

4 197.479 99.860 196.147 94.396 76 .732 783.978

5 194.612 94.240 197.186 97.771 786.241 771.571

1 186.505 81.241 198.300 87.311 710.289 749.671

2 176.188 89.968 81.780 79.731 731.985 741.325

1 199.356 87.775 207.198 101.803 748.141 771.557

2 222.589 105.058 96.074 90.785 80 .007 942.577

3 181.161 94.362 148.799 78.682 617.203 695.000

4 215.118 100.450 158.172 79.628 750.937 839.526

5 191.211 102.290 163.134 79.661 725.107 704.289

1 196.775 98.926 191.427 91.397 676. 87 806.089

2 2 8 733 94.497 88 «'7 83.249 702.543 764.559

1 189.423 103.108 176.834 85.740 787.894 783.972 2 203.993 104.8 1 242.863 100.905 744.233 775.926

1 200.471 95.865 198.445 89.203 766.594 768.276

2 202 665 90.224 209.309 97.688 728.383 781 .862

3 204.180 101 .425 203.688 92.536 677.014 860.784

4 199.399 86.145 187.369 82.171 719.400 701 .285

1 197.465 92.884 200.642 95.198 736.030 826.979

2 190.613 93.842 183.426 97.415 744.250 800.504

3 199.096 96.602 193.312 96.739 746.794 776.233

4 179.140 87.981 197.976 93.276 691 .001 866.107

1 199.248 95.517 205.052 89.399 718. 52 830.034

217.882 94.943 190.257 89.531 720.014 762.305

1 200.190 93.715 197.387 100.663 748.026 831 .065

2 213.258 94.615 190.476 91 .976 762.683 800.202

1 185.724 96.274 187.941 94.235 707.147 798.423

2 217.069 101 477 91 .859 96.905 823.232 803.7 6

3 213.982 95.430 181 .301 98.054 700.987 770.842

4 199 874 92.896 72.759 92.304 735.885 814.102

1 182.741 98.602 185.285 95.342 715.912 774.003

2 182.816 90.468 216.778 95.733 747.020 861 .042

1 179.231 95.177 187.040 88.321 748.849 740.018

2 198.872 89.425 194.280 92.437 758.636 851 .201

Mean 199.03 95.50 192.37 93.33 752.12 796.75

Sid Dev 11.02 5.62 5.76 7.07 51.25 50.69

CV 5.53 5.83 8.19 7.57 6.81 6.36

[00126] ANALYTICAL SENSITIVITY (DETECTION LIMITS)

[80127] Limit of Detection (LOD): Limit of deiection (LOD) was performed by taking a low pool (containing all analyt.es), then serially diluting (1 :2.) down to the lowest observable level. Assuming the linearity were to continue below the level of quantitation (LOQ), the values below would be the lowest quantifiable concentrations. This experiment was performed over 5 days.

Tamoxifen - 0.59 ng/mL

N-Desmefhyi Tamoxifen = 0.59 ng/mL

4' -Hydroxy Tamoxifen = 0.1 ng mL

N-DesmethyI-4' -Hydroxy Tamoxifen = 0.5 ng/mL

4-Hydroxy Tamoxifen - 0.1 ng/mL

N-Desniethyl-4-HydiOxy Tamoxifen (Endoxifen) = 0.15 ng/mL [8(5128] Limit of Quantitation (LOQ): The acceptability criteria for the LOQ is defined as the lowest concentration at which CV<20%. To determine the LOQ, a mid-level standard was diluted down serially 1 :2.

Tamoxifen ^;;; 1.5 ng/mL

N-Desmethyl Tamoxifen = 1.5 ng/mL

4'-Hydroxy Tamoxifen = 0.4 ng/mL

N-Desmethyl-4'-Hydroxy Tamoxifen = 0.4 ng/mL

4 -Hydroxy Tamoxifen = 0.2 ng/mL

-Desmethy]-4~Hydroxy T amoxifen (Endoxifen) = 0.4 ng/mL

[80129] ACCURACY

[88130] Recoven⁷ of Known Standards: Serum was spiked with all analytes to specific concentrations, extracted, and then analyzed in triplicate. Ail mixes were spiked to cover the linear and therapeutic range of each analyte.

-ΏΜ Tamoxifen 1000 885.57 857.60 932.56 891.92 37.88 4 25 Tamoxifen 1000 908.44 870.80 924.72 63.63 6.88 92.47

!V!ean: 92.81

[80131] INTERFERENCE STUDY

[8(5132] Acceptability criteria: The difference due to a potential interfering substance should be <TEa/4 to be considered acceptable

[80133] Hemolysis Interference: Low and high pools were spiked with a hemolyzed RBCs at low, medium, and high concentrations. Samples were extracted in quadruplicate. Hemolysis showed no interference with tamoxifen or measured metabolites. However, due to difficulty with filtration from moderate and high hemolysis samples, only mildly hemolyzed samples should be accepted.

[8(5134] Lipemia Interference: Low and high pools were spiked with a lipemic samples at low, medium, and high concentrations. Samples were extracted in quadruplicate. Lipemic samples showed no interference with tamoxifen or sneasured metabolites.

[80135] Bilirubin Interference: Low and high pools were spiked with a bilirubin at low, medium, and high concentrations. Samples were extracted in quadruplicate. Bilirubin spiked samples showed no interference with tamoxifen omseasured metabolites.

Example 3: Validation of norendoxifen assay

[00136] This report contains a detailed summary of the validation for Norendoxifen by LC/MS/MS. The assay is a laboratory-developed test.

[80137] Norendoxifen is extracted from serum using protein precipitation, followed by filtration. Analysis and quantitation is then performed by LC/MS/MS.

[80138] Kit or Reagents

[00139] Precision Study for Laboratory Developed ^'Tests (LDT)

[60140] Within Run Precision:Low, medium, and high controls (ng/roL) were analyzed (10) within one ran. All QC fell within acceptability criteria (<20% CV).

10 8.76 92.88 172.45

mean 8.98 96.03 180.10

std dev 0.93 8.03 18.38

%cv 10.30 8.36 10.20

[00141] Total Precision: Total precision was based on all QC run on all assays during the validation process. Acceptability was based on < 20% CV. All QC fell within this criteria.

[8(5142] Limit of Detection (LOD): Norendoxiien === 1.2 ng/mL

[80143] Limit of Quantitation (LOQ): Norendoxiie 1.2 ng/mL

[80144] ACCURACY:

[00145] Recovery of Known Standards: Serum was spiked with all anaiytes to specific concentrations, extracted, and then analyzed in quadruplicate. All mixes were spiked to cover the linear and therapeutic range of each a alyte.

Ca!ciiSs ted Concentrations (ng/mL)

Exp.

Mix Cone 1 2 3 4 iV!ean Sid Dev CV %Recov

10.00 10.67 10.53 10 27 9.04 0 3 0.75 7.37 101 .26

2 20.00 25.49 21.84 19.60 21.83 22.19 2.44 1 1.01 1 10.95

3 1 10.00 108.48 1 10.16 1 17.29 102.75 109.67 5.99 5.46 99.70

4 50.00 150.74 154.81 146.46 164.19 154.05 7.57 4.91 02.70

5 200.00 202.59 185.39 192.42 1 0.60 187.75 13.44 7.16 93.87

6 40.00 40.40 41.99 38 88 39.59 40 22 1.33 3.32 100.54

[88146] INTERFERENCE STUDY

[00147] Acceptability criteria: The difference due to a potential interfering substance should be <TEa/4 to be considered acceptable

[88148] Hemolysis Interference: Low and high pools were spiked with a hemolyzed RBC's at low, medium, and high concentrations. Samples were extracted in quadruplicate. Hemolysis showed no interference with norendoxiien. However, due to difficulty with filtration from moderate and high hemolyzed samples, only mildly hemolyzed samples should be accepted. High Poo! 101.58 112.14 114.22

[80149] Lipeniia Interference: Low and high pools were spiked with a lipemic samples at low, medium, and high concentrations. Samples were extracted in quadruplicate. Lipemic samples showed no interference with norendoxifen. Recoveiy of Known Standards: Seram was spiked with all analytes to specific concentrations, extracted, and then analyzed in quadruplicate. All mixes were spiked to co ver the linear and therapeutic range of each analyte.

[80150] Bilirubin Interference: Low and high pools were spiked with a bilirubin at low, medium, and high concentrations. Samples were extracted in quadruplicate. Bilirubin spik samples showed no interference with norendoxifen.

Example 4: Clinical quantitation and response study

[8(5151] The standard operating protocol of Examples 1-3 were used to quantitate tamoxifen and its metabolites in pat ent samples and were correlated to tamoxifen response.

[80152] Norendoxifen quantitation and response in patient samples.

20564 2.38 27537 14983 7

20573 1.63 19951 14611 5

20581 3.33 34700 1426339

20601 3.22 37814 1599850

20647 4.45 46786 1490262

20611 3.25 33755 4 5658

20689 4.36 44265 1 36705

20704 4.62 47340 1458681

26616 0.48 9695 1508141

26690 1.03 14558 1475 91

26756 1.25 16288 1447066

26777 202 20735 1287689

26801 1.70 18633 1323903

26826 2.12 16022 959275

26837 0.24 5498 1 5449

26853 2.51 18098 9447 9

26864 1.13 867 822666

26878 2.40 14663 794035

26939 1.38 9809 812995

26952 1.23 9836 886924

26958 Q.95 6946 744284

26978 167 12349 887541

27009 1.15 8821 828349

2701 2.49 16676 876309

27022 1.23 7675 689142

27045 1.34 8099 686202

27067 -016 1477 6 5842

27080 0.19 3606 7857 7

27084 0.31 4276 798627

274 3 1.04 46 1 465635

27446 -0.32 883 630981

27482 133 7572 6434 0

27526 2.78 12639 606397

27539 1.72 8159 574207

30002 0.26 2344 466484

30031 0.89 4453 495349

30043 051 3321 505290

30079 3.53 12938 505353

30105 1.54 9701 743460

301 6 0.92 6705 731395

30127 1.90 9658 629546

6420 041 64 5 1070269

6482 1.61 15116 1 21187

6497 1.40 14181 1162006

6510 0.73 6127 767435

6521 1.68 9575 687725

6535 103 8685 882954

6549 354 25094 978851

1 1.22 11722 1062595

2 2.22 18544 069320

3 461 36683 1 32746

4 17.07 117804 10566 2

5 1 .63 90103 1 72890

6 23.61 1 89 1 1 66491

7 60.70 453821 1145572

8 9924 751764 1 430 7

9 119.62 930278 1 63094

10 172.94 298434 1096393

11 238.57 1803403 1071971 12 304.02 2532623 1147835

Low1 8.83 61255 1036897

Low2 10.52 78951 1131502

Low3 11.77 58654 754843

Med1 1 2.78 786435 046058

Med2 105.19 837224 1 97875

(v1ed3 S7.48 488198 756299

HigM 203.97 1418622 1001608

High2 218.97 ■603009 1047207

High3 215.75 797944 629832

[00153] 2"^" set of data on norendoxifen

25961 38335 315946 0.020 -10.194

26081 48157 246034 0,033 1.379

26155 52963 746052 0.012 -18.043

26206 26548 1871 J? 0.024 -6.995

2Θ226 43686 258528 0.028 ^■2.778

27064 48415 95754 0.041 9.386

27106 142236 953374 0.025 -5.857

27135 64096 643672 0.017 -13.584

27186 15662 159454 0.016 -13.796

27224 28810 253 63 0.019 -11.367

27229 241 6 193237 0.021 -9.654

QC High 1 150506114043 2019214 1405526 0.239 189.933

QC Low 1 150506112054 346367 391580 0.041 9.630

QC Med 1 1505061 13049 1144208 1372839 0.139 99.309

QC High 2 295558 291585 0.169 126.576

QC High 3 616664 396020 0.260 207.821

QC Low 2 03046 3723 0 0.046 13.949

QC Low 3 59735 25S337 0.038 6.751

QC Med 2 325974 367929 0.148 107.274

QC Med 3 234615 392400 0.100 63.396

Sid 1 15050609 :206 295660 1439901 0.034 2.870

Std 2 150506092201 256587 1378734 0.031 -0. 17

Std 3 150506093155 285577 1349969 0.035 3.834

Sid 4 150506094149 415667 1443768 0.048 15.673

Std 5 150506095143 317257 315001 0.040 8.445

Sid 6 150506100138 477087 361340 0.058 25.345

Std 7 15050610 34 843486 1415920 0.099 63.062

Std 8 150506102 28 1055968 323738 0.133 93.879

Sid 9 150506103122 1310733 1333882 0.164 21.899

Std 10 150506104117 86 99 339576 0.232 182.904

Sid 11 1505061051 2398059 1352704 0.295 239.644

Std 12 150506 0106 2913618 134 519 0.362 297.950

[8(5154] Endoxifei^'i quantitation and response in patient samples.

266 9 Q.34 114 15081

26690 0.34 134 1475191

26756 1.90 34462 1447066

26777 3.36 59301 1287689

26801 5.40 102120 1323903

26826 6.68 92705 959275

26837 0.36 514 1 15449

26853 7.38 101415 944719

26864 7.57 90589 822666

26878 10.16 118857 794035

26936 2.46 26351 812995

26952 3 78 46504 886924

26958 0.34 05 744284

26978 4.26 5311 1 887541

27009 5.99 71343 828349

27011 Q.66 4334 876309

27022 6.65 66263 689142

27045 1.26 9665 686202

27067 0.94 5715 615842

27080 1.36 12324 785747

27084 1.42 13202 798627

274 3 NA MF 4656. it?

27446 1.29 92 1 630981

27482 2.88 24964 6434 0

27526 7.75 68442 606397

27539 5.66 4655ti 574207

3002 0 35 81 466484

30031 1.10 5772 495349

30043 2.54 16964 505290

30079 4.20 297 2 505353

30105 4.77 50235 743460

301 6 5 34 55768 731395

30127 6.71 61 46 629546

6420 0.41 1270 1070269

6482 2.62 39100 1 21187

6497 4.72 77527 1 62006

6510 4 39 47391 767435

6521 3.00 27958 687725

6535 7.94 102261 882954

6549 10.80 155921 978851

Std 1 1 13 12801 1062595

Std 2 2.69 38395 1069320

Std 3 4.75 7621 1 1132746

Std 4 14.64 230 6 1056612

Std 5 12.13 210594 1 72890

Std 6 23 81 4169 0 1 66491

Std 7 59.39 030203 1 45572

Std 8 94.50 638868 11430 7

Std 9 117.78 2079955 1 63094

Std 10 177.17 2952223 1096393

Std 11 248 84 4056331 1071971

Std 12 298 59 5213065 1 47835

LowQC 1 10.20 155750 1036897

LowQC 2 10.70 78553 1 31502

LowQC 3 8 24 90814 754843 edQC 1 93.72 1487561 1046058

MedQC 2 87.15 583496 1 97875

MedQC 3 83.55 958371 756299

HighQC 1 200.24 3048922 1001608 HighQC_2 j 200.35 [ 3189428 1047207 j

HighQC_3 j 206.53 j 1663579 529832 j

[00155] 4-hydroxy Tamoxifen quantitation and response in patient samples.

30079 0.71 239 7 1457036

30105 0.70 34945 2179008

30116 0.75 36168 2049184

30127 0.85 40172 1967785

6420 0.21 5225 2882321

6482 0.65 47148 3221437

6497 0.87 63944 3016193

6510 0.52 23933 2193645

6521 0.51 21842 2078718

6535 1.02 58001 2272084

6549 1.10 78339 2829487

Std 1 0.86 63333 3061651

Std 2 1.60 126439 2979412

Std 3 3.09 273498 3183743

Sid 4 10.22 857888 29 4286

Sid 5 8.14 781525 3347055

Std 6 14.99 1460920 3367273

Std 7 37.62 3585531 3271921

Std 8 67.06 6043897 3087672

Std 9 79.79 7391602 317 813

Std 10 121.24 11120485 3136830

Std 1 162.00 14892020 3141049

Std 12 197.45 18470067 3194201

LowQC 1 2.60 98332 2764071

LowQC 2 2.40 2 58 3282140

LowQC 3 2.46 24759 1845395

edQC 1 42.35 3760367 3046944

edQC 2 45.77 4055530 3039643

MedQC 3 44.83 2330598 1783433

HighQC 1 98.45 79009 3 2746235

HighQC 2 95.55 8331858 2984061

HighQC 3 96.85 4580837 16 8676

[00156] N-Desmetbyl-4' -Hydroxy quantitation and response in patient samples.

Hk-ihQC 1 219.32 16710705 1762852

HighQC_2 214.74 18007527 1940796

157] 4'-Hydroxy Tamoxifen quantitation and response in patient samples.

4'-H droxy

Tamoxifen

iSTD

Sample !D Caicii!stsd Cone (ng/mL) Response Ares

8768 0.24 3363 2172088

18788 1.19 28281 1916250

■ 8824 1.81 48728 2080977

8837 2.83 62234 1655472

8865 3.33 93441 2096079

8873 2.95 85399 2172978

18885 2.59 133675 3894668

20434 0.18 28 1 4045035

20478 0.19 3758 5065428

20484 1.06 48506 3768254

20511 0.95 44050 3859077

20515 1.90 93981 3806194

20564 2.61 ■35079 3902896

20573 2.17 04 72 3669717

20581 2.65 133250 3788223

20601 2.46 140885 4336626

20647 4.14 210537 3761489

20661 3.71 ■86268 37284 8

20689 3.44 80034 3899111

20704 3.68 1 1393 3871001

26619 0.20 3694 4050702

26690 0.24 5417 3832047

26756 0.80 34425 3712870

26777 1.19 48068 3249380

26801 1.83 66555 2809186

26826 1.78 54590 2375921

26837 0.20 2306 2467253

26853 1.86 54910 2272552

26864 1.79 44228 1 8046

26878 1.60 42925 2094936

26936 1.27 34702 2185824

26952 1.31 39922 2433167

26958 NA NF 2339037

26978 1.22 32938 2163677

27009 1.79 52321 2255996

27011 0.76 18543 2137153

27022 1.68 45719 2117327

27045 1.36 30371 1 79913

27067 2.09 48462 1770196

27080 1.73 38821 1737223

27084 1.89 39706 1622586

27413 0.26 2863 1567595

27446 0.78 14009 1553585

27482 1.30 27446 1686572

27526 2.44 48500 1505217

27539 1.98 34937 1353173

3002 0.23 1581 1130425

30031 0.39 4408 1249506 30043 1.13 20090 1446682

30079 1.58 24236 120 364

30105 1.32 35388 2136332

301 16 0.81 18620 1974717

30127 1.31 30533 1863767

6420 0.17 1208 2562866

6482 0.86 30040 2947532

6497 1.13 35827 2567403

6510 0.62 12846 1902106

6521 0.66 5265 2072439

6535 1.74 46483 2067649

6549 2.09 71871 2626700

Std 1 0.83 26072 2692512

Std 2 1.63 57494 2747701

Sid 3 3. 5 20736 2866766

Std 4 10.27 378179 2673535

Std 5 8.41 344843 2987180

Sid 6 14.49 607841 3035767

Std 7 36.08 1504777 3007474

Sid 8 66.95 2652805 2860540

Sid 9 81.81 3229939 2852941

Std 10 122.36 4801896 2844972

Sid 11 59.35 6128640 2797026

Sid 12 98.29 7827883 2880957

LowQC 1 2.13 73604 2637536

LowQC 2 2.20 79203 2747045

LowQC 3 2.20 570 9 1978114

edQC_.,.1 37.03 1384891 2696422

MedQC 2 39.46 1535649 2805856

edQC 3 37.65 837484 16039 5

HighQC 1 98.68 3324791 2437845

HighQC 2 98.97 3758231 2747447

HighQC_3 99.99 2320954 1679577

[80158] 2" set of data on 4 '-hydroxy tamoxifen

4'-Hydroxy Tairioxifen

Calculated Response

Filename Cone Response iSTD Response Ratio 9066 0.303 22269 4453051 0.001

19142 1.579 9 26 4 23038 0.004

19157 1.838 112491 4252834 0.004 9175 3.4 9 215656 4439146 0.008

19183 1.895 110049 4037559 0.005 9198 2.053 121419 4 20446 0.005 208 1.938 9044 4274660 0.005 9278 6.753 391621 4 07533 0.016 9306 6.685 283075 2998975 0.016

19309 1.328 5 548 2667301 0.003 9324 5.933 159394 1901442 0.014 360 6.746 108720 1 41453 0.016 9384 3.972 74081 13 5036 0 009 9393 5.312 9 931 1223801 0.013

19397 1.563 20063 887271 0.004 94 9 2.243 79687 2480872 0.005 443 1.866 33589 1251374 0.004 9461 2.490 35518 998353 0 006 9500 2.593 28876 779966 0.006

[00159] N-Desmetbyl Tamoxifen quantitation and response in patient samples.

N-Des methyl Tamoxifen

ISTD

Sample iD Caicuiated Cone frsg/mL) Response Area 8768 0.45 9391 7013663

■ 8788 4.72 997637 5658276

18824 6.28 1289400 5370746 8837 10.69 2081686 4945768

18865 13.75 3224539 5904185

18873 16.85 3287990 4885497

18885 0.42 6162583 503/937

20434 0.44 13754 14973184

20478 0.43 11994 19567366

20484 3.20 1640706 14365887

2051 3.53 1826641 ■4327269

20515 6.01 3287099 14345893

20564 7.67 4371978 14704733

20573 7.68 4103602 13787594

20581 7.93 4120139 13386871

20601 9.88 5544877 ■4310077

20647 7.73 9200 62 2971400

20661 16.29 9061736 13940923

20689 14.58 7719066 13305153

20704 16.47 871 935 13260540

26619 0.42 2117 ■1009897

26690 0.41 476 3027769

26756 2.22 863574 1 677656

26777 3.32 1080841 9084409

26801 5.08 1508009 7900022

26826 5.31 1381666 6897386

26837 0.43 3833 6334347

26853 5.69 1098479 508 545

26864 6.05 1 179428 5108144

26878 6.60 1541585 6084624

26936 3.81 836363 6017575

26952 3.48 913220 7282434

26958 0.43 3622 5877387

26978 4.29 1042266 6573581

27009 6.17 1529707 6486246

2701 2.03 380969 5767996

27022 5.78 236685 5625 16

27045 3.84 739878 5267957

27067 4.82 1030933 5713837

27080 5.66 1169780 5447403

27084 7.11 1364187 4971917

27413 0.42 1 08 4253160

27446 1.98 324056 5043427

27482 3.77 601493 4375000

27526 8.09 1379832 4387718

27539 6.35 1054868 4342974

3002 0.42 646 3952111

30031 1.91 235647 3844766

30043 3.87 551445 3901710

30079 5.32 748541 3728783

30105 6.47 1529920 6170754

30116 7.07 1516 84 5559182

30127 8.18 836861 5779548

6420 0.60 65522 8832583

6482 4.04 1193657 8036794 6497 6.87 2044396 7727684

6510 5.49 336093 6433864

6521 3.69 886207 6614396

6535 9.40 2555509 6943069

6549 11.01 3043467 7012004

Std 1 5.31 2327253 1 617390

Std 2 9.97 47080 4 12033677

Sid 3 19.38 9375981 12070965

Std 4 61.49 27754533 ■1094301

Std 5 49.86 25321 26 1 503328

Std 6 00.46 49441618 12062805

Std 7 237.72 12220 199 12562079

Std 8 399.25 210842807 12886373

Std 9 527.12 256541351 1 865614

Std 10 744.46 358614050 1 729628

Std 11 986.35 456157172 1 246764

Std 12 1256.13 543500909 10507976

LowQC 1 41.15 ■7191778 ■0304237

LowQC 2 40.50 _h 15299 5 9318490

LowQC 3 41.88 8361785 4924263 edQC 1 184.96 82256696 10876 49 edQC 2 176.73 62369080 8631542

MedQC 3 209.89 42232054 4918806

HighQC 1 750.34 _h 301780826 9793035

HighQC 2 724.44 245346920 8247495

HighQC 3 735.41 155798565 5158860

[8(51 0] 2 set data on N-desmethyl tamoxifen

20188 8.153 1243431 3440186 0.060

20205 8.805 2379243 6120056 0.065

21436 -0.161 82557 6275244 0.002

21546 5.755 1033811 4191694 0.043

21564 11.340 227651 1 4599180 0.082

2 603 8.428 1177190 3 564S9 0.062

2 630 13.598 1742557 2955369 0 098

21640 11.957 1582178 3037601 0.087

21653 1 1.957 1 155197 2217912 0.087

23320 2.693 31 766 2394849 0.022

23339 3.836 487883 2701690 0.030

23359 6.647 782831 2623922 0 050

23390 5.517 428388 1706743 0.042

23413 7.243 5505399 17027960 0.054

23435 4.842 378474 1699292 0.037

23438 4.856 245223 10982 7 0.037

25961 -0.352 17773 3444979 0.001

26081 -0.428 4928 2536463 0.000

26155 -0.435 3438 2053149 0.000

2Θ206 -0.421 4116 1814687 0.000

26226 -0.300 2 48 2901877 0.001

27064 -0.439 2871 1 35606 0 000

27106 4.249 1742409 8806092 0.033

27135 4.598 555198 2612748 0.035

27186 6.489 517740 1774796 0.049

27224 9.361 728755 1768581 0.069

27229 7.278 668537 2058499 0 054

QC High 1 1505061 1 043 660.452 435185890 15447019 4.695

QC Low 1 150506112054 41.893 2827 647 15914440 0.296

QC Med 1 150506113043 176.768 1 0777325 14652088 ! .243

QC_High_2 666.689 82150225 2888204 4.741

QC High 3 750.3 0 971 71 3027264 5 347

QC Low 2 59.321 7953162 3 70574 0.418

QC Low 3 42.049 3274694 1836588 0.297

QC Med 2 188. 11 27973649 3523808 ■ .323

QC Med 3 207.865 23539310 2682660 1.462

Std 1 150506091206 4.7 5 3382129 15558800 0 036

Std 2 150506092201 10.198 6944239 15530643 0.075

Sid 3 150506093155 20.520 131 13426 14905147 0.147

Std 4 150506094149 61.006 4 603966 16 30492 0.430

Sid 5 150506095143 52.335 33708040 15218576 0.369

Std 6 150506100138 00.981 6483 80S 15215929 0 710

Std 7 15050610 134 239.435 164606137 16277076 1.685

Sid 8 150506102128 400.008 25416541 14991752 2.826

Std 9 150506103122 512.940 3 5283074 14461814 3.633

Std 10 1505061041 7 751.782 473125498 14719297 5.357

Std 1 15050610511 989.021 602805977 1 168882 7 091

Std 12 150506110106 1254.549 755234278 13899320 9.056

[80161] Tamoxifen uantitation and response in patieni samples.

18865 134.03 4231612 6064968

18873 125.15 4035948 6196900

18885 54.09 5012052 1784 365

20434 0.25 29490 17918878

20478 0.05 15176 24669280

20484 39.16 3465206 170389/3

2051 1 51 .28 4521960 16978931

20515 58.91 5194907 169786 2

20564 62.95 5586963 17086731

20573 7 .94 6062564 ■6221260

20581 58.71 4699760 15412475

20601 78 44 6699955 164367 7

20647 137.89 10249638 14278153

20661 106.10 9245452 16755833

20689 90.43 74552 8 ■5860377

20704 1 16.51 9830326 16218100

26619 0.07 10074 14455436

26690 0.18 20386 15879616

26756 34.95 2383129 13131665

2Θ777 44.56 2534522 ■0951927

26801 63.56 2682884 8 25969

26826 48 06 1933458 7746196

26837 0.29 12406 667 459

26853 54.28 1547378 5488947

2Θ864 55.06 ■556148 5441919

26878 41 .41 1545348 7 85485

26936 40 27 1394728 66690 2

26952 27.71 1 107256 7694108

26958 0.33 16221 7897622

2Θ978 19.13 754743 7589060

27009 50.25 927906 7387061

2701 1 36 56 1358366 7154478

27022 49.21 1655736 6478414

27045 48.06 561893 6258393

27067 49.26 ■517871 5933913

27080 48.76 1474533 5822771

27084 40 5 1262719 6055936

27413 0.25 8576 5300227

27446 26.64 793351 5733720

27482 38.93 ■066943 5277298

27526 65.81 659523 4854 08

27539 66 06 1601415 4666695

3002 0.26 7653 4493100

30031 12.60 296745 4522346

30043 30.38 776495 4921844

30079 33.53 81 30 4658023

30105 26 33 941949 6886006

301 16 20.53 705407 661 1936

30127 36.97 165680 6071292

6420 0.61 369 5 10553272

6482 18.90 99 389 1009 984

6497 33 76 1545743 8815779

6510 18.08 687863 7318004

6521 4.28 572749 7707224

6535 56.47 2235005 7619942

6549 60.89 2473508 7820955

Std 1 5.07 368746 13870144

Std 2 9.87 743436 14447818

Std 3 20.91 1577523 14518514 Sid 4 61 .61 4389679 13716703

Std 5 50.98 3886987 14681961

Std 6 98.50 7499571 14642835

Std 7 232.22 18185150 14991747

Std 8 401 .17 31914385 ■5135753

Sid 9 504.67 38342009 14399860

Sid 10 761 .87 53303371 13136206

Std 1 1 028.75 70717814 12781973

Std 2 1221 .75 91208237 13784726

LowQC 1 37.88 2612589 13281768

LowQC 2 37.79 2103909 10721385

LowQC 3 39.37 1299297 6355004 edQC 1 195.63 12368756 121 19500

MedQC 2 208.87 9917798 9097656

MedQC 3 192.21 6176283 6 60378

HighQC 1 753.79 47482016 11830479

HighQC 2 783.21 37796034 9053718

HighQC 3 792.09 227321 14 5382465

[80162] 2" set of data on tamoxifen

21653 50.235 829879 2956721 0.047

23320 40.8 4 567630 24818 9 0.038

23339 56.680 920885 291 1936 0.053

23359 82.388 947509 2067360 0.078

23390 56.566 728329 2307661 0.053

23413 02.246 4437 77 1 788 :0 0.058

23435 39.294 493294 2238856 0.037

23438 38.475 344868 1597971 0.036

25961 0.037 17722 4 42657 0.001

26081 ■0.254 6716 2510216 0.000

26155 -0.439 4001 24 3366 0.000

26206 0.050 1 022 2534035 0 001

26226 -0.200 14415 4846489 0.000

27064 -0.023 9656 2445806 0.001

27106 42.931 2980808 12399985 0.040

27 35 50.710 9 7164 32375 6 0.047

27186 52.669 544080 1849951 0.049

27224 69.349 849916 2200587 0.064

27229 43.288 561047 23 4994 0.040

QC High 1 150506114043 665.297 75125747 13977315 0.627

QC Low 1 1505061 2054 42.707 4685691 19592959 0.040

QC Med 1 150506113049 91.494 933 542 18 : 58006 0 177

QC High 2 702.467 14501479 3646902 0.663

QC High 3 693.744 14196581 3616342 0.654

QC Low 2 56.305 1102329 3508672 0.052

QC Low 3 44.334 546776 2203665 0.041

QC Med 2 95.004 4267239 3935737 0 181

QC Med 3 231 .177 3406054 2647533 0.214

Sid 1 150506031206 4.730 590077 19625344 0.005

Std 2 150506092201 10.057 1163202 13597467 0.010

Sid 3 150506093155 20.691 2175560 18458380 0.020

Sid 4 5050609 49 57.908 6569960 20338991 0 054

Std 5 150506095143 52.677 5527146 18790321 0.049

Sid 6 150506100138 104.641 ■0935888 18805047 0.097

Std 7 150506101134 240.132 26496831 19823070 0.223

Sid 8 150506102128 403.963 42234460 18680230 0.377

Std 9 150506103122 521.039 53364854 18220106 0 488

Std 10 150506104117 745.879 78023680 18448605 0.705

Sid 11 1505061051 11 957.878 98106705 1 914330 0.913

Std 12 1505061 0106 1277.8 3 136400695 18438987 .233

[80163] Patient data summary

N-DM-4'-

Sample n-DM- OH- 4-OH- 4'-OH-

!D # Tamoxifen Tamoxifen Tamoxifen Ersdoxifen ioreradoxifers Tamoxifen Tamoxifen

19068 3.701 2.065 0.766 1.391 8.564 0.332 0.303

19142 46.038 5. 93 0.916 4.316 -2.102 1 .047 1.579

19157 42.676 5.814 0.831 5.240 -5.625 1 .054 1.838

19175 70.899 15.766 2.934 26.761 -0.471 .779 3.419

19183 40.556 6.688 1.480 6.161 0.910 1.378 1.895

19198 50.601 8.157 1.345 6.282 0.341 1.2 7 2.053

19208 48.961 5.735 0.691 6.009 -2.520 0.753 1.938

19278 1 12.000 23.120 4.595 39.457 4.901 2.840 6.753

19306 09.572 27.789 5.282 37.314 3.408 3.227 6.685

19309 12.404 6.095 1.61 1 3.442 -5.207 0.765 1.328

19324 16.499 19.251 4.073 35.628 2 215 2.209 5.933

19360 1 15.255 24.238 4.243 44.038 0 95 2.461 6.746 19384 69.683 19.202 3.648 37.655 -0.326 1 .757 3.972

19393 93.2Θ4 17.748 2.821 35.239 -0.774 1 .565 5.312

19397 58.819 4.674 0.193 5.816 1 .414 1.563

19419 64.295 5.292 0. 94 7.615 -5.833 1 .386 2.243

19443 65.809 5.792 0.251 8.806 -6.791 1 .407 1.866

19461 76.288 7 933 0.467 2.942 -5.208 1 .592 2.490

19S00 76.305 9 755 0.569 15.373 -3.262 1 .61 1 2.593

19513 68.1 10 8.469 0.692 16.571 -4.032 1 .350 2.550

19539 45.479 9.135 0.760 17.872 0.679 1.142 1.572

20081 32.732 2.233 0.296 2.256 -5.750 0.447 0.586

20124 48.263 5.001 0.518 4.950 0.254 1.023 1.781

20140 68.004 5.948 0.494 7.486 1.612 0.996 1.899

20 60 64.933 7.198 0.477 8.130 -16.105 0.868 1.828

2017S 75.348 7.559 0.634 8.067 -10.658 0.645 1.554

20188 89.929 8.153 0.847 10.347 -1.477 1 .275 1.898

20205 70.704 8.805 0.660 8.673 -3.083 0.855 2.902

21436 1.476 -0.161 -0.1 9 0.085 -2.327 0.075 0.1 17

21546 48.585 5.755 0.213 1.539 -3.604 0.371 1.348

21564 95.890 1 1.340 0.705 3.078 -9.549 0.658 2.889

21603 63.763 8 428 0.655 2.686 -2.349 0.546 2.569

21630 84.1 13 13.598 1.362 4.806 -5.337 0.615 4.485

21640 82.497 1 1.957 1.328 3.512 -3.461 0.600 4.175

21653 50.235 1 1.957 1.349 3.71 -5.925 0.688 2.931

23320 40.814 2.693 0.290 0.784 -7.143 0.646 0.548

23339 56.680 3.836 -0.075 3.599 -16.431 0.9 7 1.272

23359 82.388 6.647 0.278 5.589 -7.287 1 .174 2.257

23390 56.566 5.517 0.254 4.420 -22.698 1.01 1 1.908

23413 62.246 7.243 0.584 6.938 -2.949 0.976 2.272

23435 39.294 4.842 0.390 4.922 2.446 0.571 2.232

23438 38.475 4.856 0.431 5.276 3.548 0.655 1.798

25961 0.037 -0.352 -0.108 0.072 -10.194 0.075 0.253

26081 -0.254 -0.428 -0.133 0.063 1.379 0.023 -0.023

261S5 -0.439 -0.435 -0.163 A -18.043 0.001 0.064

26206 0.050 -0.421 -0.083 0.015 -6.995 0.062 0.544

26226 -0.200 -0.300 -0.150 NA -2.778 0.054 0.210

27064 -0.023 -0.439 -0.122 0.013 9.386 0.056 0.026

27106 42.931 4.249 0.148 4.712 -5.857 1 .267 1.523

27135 50.710 4.598 0.264 5.862 -13.584 0.958 1.867

27186 52.669 6.489 0.189 7.137 -13.796 0.874 2.309

27224 69.349 9.361 0.934 14.139 -1 1.367 1.390 2.862

27229 43.288 7.278 0.562 12.816 -9.654 1 .055 2.767

Example 5: Additionai clinical quantitatio and response study

[80164] The standard operating protocol of Examples 1-3 were used to quaniitate tamoxifen and its metabolites in patient samples and were correlated to tamox fen response.

[00165] 4'-hydroxy tamoxifen Std 1 0.800 -19 0.650 1 18875 2072069 0.002 3.3 69

Sid 2 .600 4 .671 2 51081 2234410 0.004 3.3316

Std 3 3.200 2 3.866 3 113684 2168822 0.009 3.3309

Sid 4 10 000 -11 8.924 4 271360 2253046 0.020 3.3313

Sid 5 8.000 4 8.305 5 256942 2291669 0,019 3,3313

Std 6 16.000 0 15.936 6 476695 2221478 0.036 3.33 0

Sid 7 40.000 -3 38.805 7 95727 2297958 0.087 3.3309

Std 8 64.000 4 66.462 8 _h 9787 0 2229656 0.148 3.3314

Sid 9 80 000 6 84.720 9 25008 8 2216656 0.188 3.33 3

Sid 10 120.000 -10 108.549 10 3532184 2452070 0,240 3,33 3

Std 11 160.000 4 166.177 11 5013004 2292376 0.364 3.3309

Sid 12 200.000 0 199.566 12 6005444 2297913 0.436 3.3310

QC Low 1 2.000 26 2.528 1 Low 79280 2304249 0.006 3.3026

QC Low 2 2 000 11 2.212 1 Low 77290 2564284 0.005 3.3597

QC Low 3 2.000 6 2.110 1 Low 87323 3034648 0,005 3,3885

QC Med 1 40.000 1 40.300 2 Med 299131 2404628 0.090 3.3167

QC Med 2 40.000 -5 38.185 2 Med 1228576 2399191 0.085 3.3596

QC Med 3 40.000 5 42.171 2 Med ■549585 2741809 0.094 3. 888

QC High 1 100 000 1 101.497 3 High 3219275 2387664 0.225 3.3173

QC High 2 100.000 -7 92.799 3 High 30203 9 2446947 0,206 3,3600

QC High 3 100.000 1 100.902 3 High 3751385 2798463 0.223 3.3884

45301 NA NA 4.590 NA 155747 2505735 0.010 3.3310

45390 NA NA 8.253 NA 322666 2896059 0.019 3.3454

45466 NA NA 3.246 NA 27068 2883372 0.007 3.3314

45485 NA NA 0.591 NA 22294 2680691 0,001 3,3315

45569 NA NA 0.019 NA 1596 2641180 0.000 3.4171

45634 NA NA 3.033 NA 121975 2960336 0.007 3.3453

45717 NA NA 4.887 NA 1963 6 2967875 0.01 3.3457

45750 NA NA 2.940 NA 19665 2995645 0.007 3.3460 5752 NA NA 6.298 NA 255549 3001506 0,014 3,3457

45798 NA NA 2.022 NA 91673 3323059 0.005 3.3453

45800 NA NA 5.486 NA 225841 3043295 0.012 3.3453

45810 NA NA 3.438 NA 1333 5 2857926 0.008 3.3457

45825 NA NA 6.583 NA 260550 2928766 0.015 3.3453

45835 NA NA 3 962 NA 56772 2918803 0,009 3,3461

45867 NA NA 3.108 NA 123680 2929971 0.007 3.3457

45946 NA NA 5.147 NA 99901 2869775 0.012 3.3457

4Θ037 NA NA 5.643 NA 230964 3026251 0.013 3.3453

46147 NA NA 8.247 NA 335804 3016 26 0.01 3.3601

46153 NA NA 3 098 NA 19539 2841244 0,007 3,3458

46180 NA NA 3.884 NA 153272 2910985 0.009 3.3453

46213 NA NA 2.493 NA 09506 3227714 0.006 3.3603

46221 NA NA 3.541 NA 130860 2723957 0.008 3.3745

46283 NA NA 3.892 NA 38762 2629759 0.009 3.3601

46301 NA NA 5 251 NA 86491 2624655 0,012 3,3743

46428 NA NA 3.622 NA 132933 2705965 0.008 3.3746

46442 NA NA 2.922 NA 14861 2893174 0.007 3.3744

46453 NA NA 2.527 NA 99000 2879043 0.006 3.3601

46484 NA NA 3.743 NA 144129 2839205 0.008 3.3747

46518 NA NA 3 08 NA 139 0 2698427 0,007 3,3747

46541 NA NA 4.711 NA 181415 2844378 0.01 3.3740

46548 NA NA 4.970 NA 90113 2826310 0.011 3.3740

46606 NA NA 2.090 NA 76649 2689489 0.005 3.3744

46667 NA NA 10.765 NA 461374 3 77987 0.024 3.3740

46717 NA _l____NA_ 7 253 NA 304387 3 06739 0,016 3,3744

46731 NA NA 4.273 NA 182477 3152075 0.010 3.3744

46735 NA NA 2.919 NA 37705 3471886 0.007 3.3744

46749 NA NA 3.876 NA 174803 3326210 0.009 3.3884

46841 NA NA 3.195 NA 52289 3510357 0.007 3.3884 46859 NA NA 6.391 NA 308879 3575840 0.014 3.3888

46870 MA MA 3.938 NA 87297 3508553 0.009 3.3741

46896 NA NA 6.647 NA 310768 3459381 0.015 3.3744

469 5 MA MA 0.314 NA 15189 3317417 0.001 3.3884

B1121 NA NA -0.011 NA 712 3604644 0,000 3,3889

B423 NA NA 7.737 NA 286757 2744489 0.017 3.3456

B599 MA MA 5.042 NA 93874 2841340 0.01 3.3457

B614 NA NA 3.958 NA _h 161177 3004219 0.009 3.3454

B651 MA MA 3.046 NA 14124 2758319 0.007 3.3740

B702 NA NA 3.499 NA 121390 2557001 0,008 3,3748

4'-Hydroxy

■ arnoxifen

Specified % Caicuiated !STD Response

Sample iD Cone Diff Cone Level Response Response Ratio RT

Sid 1 0 800 5 0.840 1 35249 29587 0.002 3.3003

Sid 2 1.600 8 1.735 2 72861 2980562 0,004 3,3093

Std 3 3.200 -2 3.124 3 141591 3225973 0.007 3.3200

Sid 4 10.000 -5 9.538 4 424861 3 79029 0.022 3.3208

Std 5 8.000 , -Z_ 7 745 5 355442 3274330 0,018 3.3195

Sid 6 16 000 -4 15.293 6 734203 3429 3 0.036 3.3091

Sid 7 40.000 -4 38.406 7 17795 5 3315673 0,089 3,3088

Std 8 64.000 3 65.945 8 2961260 3219341 0.153 3.3187

Sid 9 80.000 3 82.622 9 3780953 3284353 0.192 3.3205

Std 10 120.000 ^■2 117.460 10 5720657 3503326 0.272 3.3206

Sid 11 160 000 163.231 11 7179921 3 73352 0.377 3.3199

Sid 12 200 000 -1 197.662 12 9562222 3497821 0.456 3.3209

OC Low 2 2.000 27 2.535 1 Low 329 7 923798 0.006 3.3201

OC Low 2 2.000 ^■2 ■ .964 1 Low 64960 2349338 0.005 3.3408

QC Low 1 2 000 3 2.061 1 Low 97498 3361269 0.005 3.3205

QC Low 3 2.000 12 2.234 1 Low 289 1 920883 0,005 3,3500

QC Med 1 40.000 -4 38.547 2 Med 1884050 3497570 0.090 3.3195

QC Med 2 40.000 -2 39.081 2 Med 566023 036456 0.091 3.3303

QC Med 2 40.000 ^■14 34.202 2 Med 182282 381263 0.080 3.34 3

QC Med 3 40 000 -7 37.337 2 Med 341288 654060 0.087 3.3406

QC High 1 100.000 -5 94.884 3 High 4598348 3480962 0,220 3,3193

QC High 2 100.000 -3 96.711 3 High 251484 929592 0.224 3.3315

QC High 2 100.000 -4 95.878 3 High 578257 433233 0.222 3.3402

QC High 3 100.000 6 105.842 3 High 892150 605869 0.245 3.3395

46917 NA NA 6.294 NA 359107 4069 11 0,015 3,3 05

46951 NA NA 6.037 NA 358297 4232608 0.014 3.3187

46960 NA NA 4.332 NA 259114 4262102 0.010 3.3205

46990 NA NA 3.019 NA 186373 4393941 0.007 3.3299

47076 MA MA 4.647 NA 286503 4393808 0.01 3.3313

47148 NA NA 3.378 NA 203309 4284871 0,008 3,3213

47204 NA NA 4.003 NA 246164 4381015 0.009 3.3207

47223 NA NA 5.265 NA 329471 4461284 0.012 3.3197

47259 NA NA 4.429 NA 280965 4520722 0.010 3.3307

47262 MA MA 2.216 NA 146133 688581 0.005 3.3298

47271 NA NA 3.533 NA 210174 4237018 0,008 3,3204

47285 NA NA 2.812 NA 167507 4239139 0.007 3.3209

47287 NA NA 4.284 NA 247990 4 24588 0.010 3.3198

47294 NA NA 6.148 NA 304761 3535140 0.014 3.3299

47296 MA MA 6.503 NA 415945 4561599 0.015 3.3196

47362 NA NA 6.575 NA 217818 2362652 0,015 3,3205

47398 NA NA 1.552 NA 56103 2564963 0.004 3.3303

47408 NA NA 4.236 NA 140137 2356955 0.010 3.3298

47429 NA NA 2.437 NA 61808 ■804049 0.006 3.32 3

47478 MA MA 6.839 NA 146298 525812 0.016 3.3309 47507 NA NA 5.091 NA 72042 008788 0.012 3.3297

47512 NA NA 5.454 NA 74965 979958 0,013 3,3307

47547 NA NA 8.056 NA 1 16192 029098 0.019 3.3290

47555 NA NA 3.667 NA 57301 1112989 0.009 3.3297

47583 NA NA 6.473 NA 86404 9520 0 0.015 3.3196

47586 NA NA 5.818 NA 76240 934334 0.014 3.3410

47626 NA NA 3.746 NA 45479 864793 0,009 3,3094

47629 MA NA 3.136 NA 29948 679734 0.007 3.3413

47633 NA NA 3.606 NA 26615 525691 0.008 3.3304

47674 NA NA 0.093 NA 847 585035 0.000 3.36 2

47696 NA NA 3.824 NA 250106 4658812 0.009 3.3297

47715 NA NA 5 750 NA 261070 3237523 0,013 3,3313

B109 NA NA 1.524 NA 14768 687552 0.004 3.3488

B124 NA NA 8.718 NA 30895 252876 0.020 3.3412

B!38 NA NA 3.821 NA 75737 ■411978 0.009 3.3510

B151 NA NA 4.594 NA 57488 891738 0.01 3.3414

B174 NA NA 2.520 NA 26797 756341 0,006 3,3405

B209 NA NA 6.388 NA 48930 546310 0.015 3.3391

B211 NA NA 3.127 NA 38515 876857 0.007 3.3300

B218 NA NA 3.539 NA 369 9 742987 0.008 3.3303

B249 NA NA 5.207 NA 21627 296085 0.012 3.3407

B348 NA NA 3 459 NA 73040 503576 0,008 3,3409

B379 NA NA 6.245 NA 28271 322831 0.015 3.3412

B416 NA NA 3.862 NA 17684 326151 0.009 3.3410

B417 NA NA 4.720 NA 176904 26710 0 0.01 3.3198

B50 NA NA 0. 11 NA 667 393861 0.000 3.3521

B77 NA NA 4 677 NA 89665 366217 0,01 3,3421

B793 NA NA 5.094 NA 21269 297647 0.012 3.3317

B875 NA NA 3.932 NA 27943 506274 0.009 3.3405

B96 NA NA 2.546 NA 22119 617916 0.006 3.3514

[80166] N-Desmethyl Tamoxifen

3

QC High 1 800.000 1 806,621 High 349559336 10739364 5,425 4.6272

3

QC High 2 800.000 -4 766,892 High 321869979 10399085 5,159 4.6699

3

QC High 3 800.000 -3 772,284 High 369290721 11848163 5,195 4.6839

45301 NA NA 26.273 NA 11841632 10945501 0.180 4.6552

45390 NA NA 39.710 NA 18654553 11477691 0.271 4.6553

45466 MA MA ■ 4.131 MA 7051102 11934357 0.098 4.6556

45485 NA NA 2. 04 NA 1235242 1839387 0.017 4.6558

45569 NA NA -0,438 NA 7606 11822707 0,000 4.6553

45634 NA NA 18.338 NA 9228298 12127381 0.127 4.6552

45717 NA NA 0.770 NA 5532916 12163713 0.076 4.6556

45750 MA MA 4.916 MA 27 7384 12460010 0.036 4.6559

45752 NA NA 23.367 NA 2033795 2478509 0.161 4.6699

45798 A NA 8,081 NA 45 4702 13045004 0,058 4.6695

45800 NA NA 12.381 NA 6600043 12691932 0.087 4.6552

45810 NA NA 21.579 NA 10724905 12022578 0.149 4.6557

45825 MA MA 33.817 MA 17048836 12292179 0.231 4.6696

45835 NA NA 11.219 NA 5770806 21 9963 0.079 4.6704

45867 NA NA 12,834 NA 6456515 11992891 0,090 4.6699

45946 NA NA 12.791 NA 6297921 1 1 36675 0.089 4.6557

46037 NA NA 10.380 NA 5360503 12208515 0.073 4.6695

4Θ147 MA MA _h 44.324 MA 21087917 11638790 _h 0.302 4.6700

46153 NA NA 3.246 NA 6744190 12151619 " 0.093 4.6700

46180 NA NA 3,893 NA 7099200 12215198 0,097 4.6696

46213 NA NA 13.928 NA 7116545 12214797 0.097 4.6702

46221 NA NA 26.106 NA 1 1555669 10748057 0.179 4.6701

4Θ283 MA MA 24.324 MA 10709139 10676283 0.167 4.6843

46301 NA NA 29.768 NA 140 3373 1455981 0.204 4.6842

46428 NA NA 9,931 NA 456381 10842547 0,070 4.6845

46442 NA NA 6.573 NA 3284257 1 1519780 0.048 4.6843

46453 NA NA 14.676 NA 7224761 11788779 0.102 4.6844

4Θ484 MA MA ■ 5.352 MA 6937804 10836949 0.107 4.6846

46518 NA NA 5.940 NA 2822979 0878008 0.043 4.6846

46541 NA NA 12,145 NA 5456620 10689596 0,085 4.6839

46548 NA NA 7.369 NA 7686125 10648745 0.120 4.6839

46606 NA NA 13.382 NA 6230069 11114932 0.093 4.6843

4Θ667 MA MA 54.075 MA 26635133 12073060 0.368 4.6839

46717 NA NA 32. 89 NA 5985951 2099953 0.220 4.6843

46731 NA NA 23,463 NA 1 1781079 12167379 0,161 4.6843

46735 NA NA 10.658 NA 6231222 13836352 0.075 4.6844

46749 NA NA 18.278 NA 10047672 13245981 0.126 4.6839

46841 MA MA 23.201 MA 12927666 13499605 0.160 4.6839

46859 NA NA 35.736 NA 20430737 0.244 4.6843

46870 NA NA 9,464 NA 5495150 3667994 0,067 4.6840

46896 NA NA 39.389 NA 22095261 13704056 0.269 4.6843

46915 NA NA 1.258 NA 945809 13492832 0.012 4.6839

B■ 121 MA MA ^■0.449 MA ■ 4423 136 086 0.000 4.6845

B 23 NA NA 33.052 NA 5825521 1670269 0.226 4.6556

B599 NA NA 5,587 NA 7789173 1988574 0,108 4.6556

B614 NA NA 14.595 NA 7250764 1 1894417 0.102 4.6553

B651 NA NA 17.086 NA 8096715 1 1398887 0.118 4.6839

B702 MA MA 20.529 MA 8668370 10203235 0.142 4.6847

N-Desmethyi

Tamoxifen

Specified % Calculated !STD Response

Sample !D Cone Diff Cone Levei Response Response Ratio RT

Sid 1 5.000 4 5.216 1 3035366 12894595 0.039 4.6451

Stc 2 0.000 5 ■ 0.513 2 6039685 13277968 0.076 4.6539 Sid 3 20.000 0 9.997 3 12196051 14385916 0.141 4.6542

Std 4 62.500 -6 58,667 4 347 3350 14166401 0,408 4.6556

Std 5 50,000 -2 48.758 5 29854945 14637029 0.340 4.6434

Sid 6 100.000 -3 96.677 6 60835111 15109059 0.671 4.6533

Std 7 250.000 ^■1 247.393 7 1504 7601 14628708 1.714 4.6430

Sid 8 400.000 5 4 8.186 8 249322031 14341070 2.898 4.6422

Sid 9 500.000 0 502,301 9 31 1783615 4926066 3,481 4.6444

Std 10 750.000 -2 736.267 10 481407377 15705917 5.109 4.6445

Sid 11 1000.000 0 1004.915 11 593251935 14160635 6.982 4.6440

Sid 12 1250.000 0 1248.606 12 768772647 14749010 8.687 4.6449

1

OC Low 2 40.000 -1 35.438 Low 5014390 3370741 0.248 4.6642

1

OC Low 2 40.000 -1 39.697 Low 8530728 5126252 0.277 4.6752

1

QC Low 1 40.000 0 40.102 Low 25849688 15378255 0.280 4.6547

1

QC Low 3 40.000 4 .700 Low 5035346 2882014 0.291 4.6636

2

QC Med 1 200.000 -6 187.687 Med 122092292 15647367 1.300 4.6539

2

QC Med 2 200.000 -22 156.276 Med 686199 3029159 1.083 4.6544

2

QC Med 2 200.000 -6 187.708 Med 9278718 1189027 1.301 4.6760

2

QC Med 3 200.000 -7 185.679 Med 14183336 1837368 1.287 4.6646

3

QC High 1 800.000 -5 756.162 High 47503 778 15088636 5.247 4.6434

3

QC High 2 800.000 -16 674.720 High 70649330 25 5953 4.680 4.6456

3

QC High 2 800.000 -8 733.6 8 High 4686051 1534366 5.090 4.6643

3

QC High 3 800.000 -6 75 .226 High 64370878 2058125 5.2 3 4.6634

46917 NA NA 26.132 NA 181 9897 6442596 0.184 4.6552

46951 NA NA 17.249 NA 121 8193 65 132 0.122 4.6630

46960 NA NA 8.233 NA 3027133 68 5396 0.129 4.6548

46990 NA NA 4,714 NA 3672603 71 2987 0,036 4.6638

47076 NA NA 17.044 NA 12399477 17092640 0.121 4.6554

47148 NA NA 0.75 NA 7738928 6653008 0.077 4.6557

47204 MA NA 21.609 NA 5138607 16552674 0.152 4.6551

47223 NA NA 26.069 NA 8575235 6896149 0.183 4.6536

47259 A NA 24,482 NA 1790 150 73 8645 0,172 4.6653

47262 NA NA 1 1.522 NA 8873081 17865097 0.083 4.6639

47271 NA NA 16.032 NA 11167382 6337683 0.1 4 4.6551

47285 NA NA 10.523 NA 6632132 14567325 0.076 4.6551

47287 NA NA 7.253 NA 4150522 2977664 0.053 4.6644

47294 NA NA 23,672 NA 10097368 0096633 0,167 4.6642

47296 NA NA 25.731 NA 18048131 16627998 0.181 4.6639

47362 NA NA 30.720 NA 9623565 7447965 0.2 5 4.6651

47398 NA NA 11.900 NA 2953091 5764235 0.085 4.6647

47408 NA NA 14.802 NA 2642033 4176803 0.105 4.6641

47429 NA NA ,723 NA 2739959 5425673 0,084 4.6662

47478 NA NA 26.901 NA 5450199 4806728 0.189 4.6650

47507 NA NA 25.265 NA 3608824 3385136 0.178 4.6642

47512 NA NA 22.956 NA 2507451 2583917 0.162 4.6653

47547 NA NA 39.779 NA 5279817 3166205 0.278 4.6629 7555 NA NA 5,859 NA 2083193 3080093 0,113 4.6742

47583 NA NA 8.888 NA 929424 2398272 0.065 4.6641

47586 NA NA 30.921 NA 3210173 2468503 0.217 4.6650

47626 NA NA ■ 8.322 NA 1864197 2394881 0.130 4.6640

47629 NA NA 5. 35 NA 450948 1943285 0.039 4.6761

47633 NA NA 12,866 NA 967726 1751989 0,092 4.6645 47674 NA NA -0.454 NA 874 16 2859 0.000 4.6644

47696 NA NA 6,209 NA 3169773 4588361 0,115 4.6641

47715 NA NA 3.470 NA 1656231 2868681 0.096 4.6758

B109 NA NA 11.432 NA 1033667 2097056 0.082 4.6825

B124 MA NA ■4.692 NA 692056 1101989 0.105 4.6754

B 38 NA NA 22.437 NA 1400162 1475589 0.158 4.6752

B 51 NA NA 21 ,416 NA 1047332 1155265 0,151 4.6656

B174 NA NA 10.349 NA 412904 921568 0.075 4.6644

B209 NA NA 14.006 NA 798577 1331885 0.100 4.6731

B211 NA NA ■ 1.346 NA 432864 884514 0.082 4.6748

B2 8 NA NA 14.9 5 NA 990100 1553714 0.106 4.6751

B249 NA NA 5.747 NA 589399 877488 0.112 4.6749

B348 NA NA 5.742 NA 1252 157 1864674 0.112 4.6755

B379 NA NA 16.374 NA 789263 1131237 0.116 4.6754

B416 NA NA ■ 3.889 NA 652860 1097759 0.099 4.6655

B4 7 NA NA 7.747 NA 4252133 5635185 0.126 4.6645

B50 NA NA -0,344 NA 10474 2062412 0,001 4.6659

B77 NA NA 13.213 NA 997904 1760839 0.094 4.6666

B793 NA NA 16.843 NA 766191 1068454 0.120 4.6764

B875 NA NA ■ 9.567 NA 1696007 2043385 0.138 4.6748

B96 NA NA 9.888 NA 952563 2220513 0.071 4.6755

[80167] Tamoxifen

3

QC High 2 800 000 -9 729-436 High 4882360Θ 12758558 QC Sample 0.638 4.9035

3

QC High 3 800 000 ^■Θ 750-663 55810349 14168310 QC Sample 0.657 4.9032

Unknown

45301 NA NA 130.875 NA 9201591 13424057 Sample 0.1 14 4.8745

Unknown

45390 NA NA 142.588 NA 10729349 14374421 Sample 0.124 4.8889

Unknown

45466 NA NA 51 .861 NA 3887923 14153276 Sample 0.046 4.8892

Unknown

45485 NA NA 2.025 NA 225794 14029301 Sample 0.003 4.8893

Unknown

45569 NA NA -0.642 NA 31725 13993172 Sample 0.000 4.9032

Unknown

45634 NA NA 79.422 NA 6087679 14568254 Sample 0.070 4.8888

Unknown

45717 NA NA 1 14.657 NA 8769809 14590327 Sample 0.100 4.8892

Unknown

45750 NA NA 26.715 NA 2100131 14567323 Sample 0.024 4.8895

Unknown

45752 NA NA 103.765 NA 7994477 14684389 Sample 0.091 4.8892

Unknown

45798 NA NA 35.068 NA 2844990 15171845 Sample 0.031 4.8888

Unknown

45800 NA NA 50.405 NA 3867320 14476922 Sample 0.045 4.8888

Unknown

45810 NA NA 1 15.360 NA 8718130 14416627 Sample 0.101 4.8893

Unknown

45825 NA NA 179.654 NA 13173726 14022261 Sample 0.157 4.8889

Unknown

45835 NA NA 1 16.025 NA 8544018 14048402 Sample 0.101 4.8896

Unknown

45867 NA NA 90.584 NA 6772504 14231363 Sample 0.079 4.8892

Unknown

45946 NA NA 68.180 NA 5068384 14099973 Sample 0.060 4.8893

Unknown

46037 NA NA 122.733 NA 9267700 1441 1275 Sample 0.107 4.8888

Unknown

46147 NA NA 245.606 NA 18005272 14028 74 Sample 0.214 4.8892

Unknown

46153 NA NA 64.050 NA 4709356 13932869 Sample 0.056 4.8893

Unknown

46180 NA NA 45.657 NA 3482346 14361051 Sample 0.040 4.8888

Unknown

46213 NA NA 39.700 NA 3002720 14193252 Sample 0.035 4.8894

Unknown

46221 NA NA 60.8 7 NA 4132298 12864807 Sample 0.054 4.9037

Unknown

46283 NA NA 106.601 NA 7203546 12882631 Sample 0.093 4.9036

Unknown

46301 NA NA 175.670 NA 12247590 13331077 Sample 0.153 4.9035

Unknown

46428 NA NA 102.971 NA 7021 703 12996 79 Sample 0.090 4.9037

Unknown

46442 NA NA 34.971 NA 26 2135 13967304 Sample 0.031 4.9036

Unknown

46453 NA NA 76.528 NA 5324388 13217410 Sample 0.067 4.9036

Unknown

46484 NA NA 134.492 NA 9125489 12957248 Sample 0.1 17 4.9038

Unknown

46518 NA NA 32.697 NA 2256501 12878575 Sample 0.029 4.9182

Unknown

46541 NA NA 77.359 NA 4843270 1 1895464 Sample 0.068 4.9175

Unknown

46548 NA NA 85.387 NA 5732477 12770877 Sample 0.075 4.9032

Unknown

46606 NA NA 59.848 NA 4199528 13282068 Sample 0.053 4.9179

Unknown

46667 NA NA 135.598 NA 10414841 14668087 Sample 0.1 18 4.9032

Unknown

46717 NA NA 182.1 12 NA 14165239 14874821 Sample 0.159 4.9036

46731 MA NA 145.655 NA 10828856 14203833 Unknown 0. 27 4.9036 Sample

Unknown

46735 NA NA 60.560 NA 5245978 16400049 Sample 0 053 4 9180

Unknown

46749 NA NA 97.135 NA 8098485 15881 33 Sample 0 085 4 9032

Unknown

46841 MA NA 91.310 NA 7725533 16 06 90 Sample 0 080 4 9032

Unknown

46859 MA NA 129.621 NA 1 98473 16494335 Sample 0 113 4 9036

Unknown

46870 MA NA 94.153 NA 7837005 i 5850306 Sample 0 082 4 9176

Unknown

46896 MA NA 152.754 NA 13132709 16429 50 Sample 0 133 4 9035

Unknown

469 5 MA NA 2.000 NA 257251 16 13 6 Sample 0 003 4 9032

Unknown

B1121 MA NA -0.601 NA 397 4 16006905 Sample 0 000 4 9181

Unknown

B 3 MA NA 140.264 NA 10248102 13955854 Sample 0 122 4 8748

Unknown

B599 MA NA 68.107 NA 5070892 14121922 Sample 0 060 4 8892

Unknown

B6 4 MA NA 74.326 NA 5545621 14 69095 Sample 0 065 4 8889

Unknown

B651 MA NA 63.727 NA 4449989 13231237 Sample 0 056 4 9175

Unknown

B702 MA NA 67.581 NA 43 5968 12 11629 Sample 0 059 4 9040

Calculated

Sample !D Specified Cone Cone % Diff Level Response !STD Response Response Ratio RT

Std_1 5.000 5.166 3 1 5 8618 5827044 0.005 4.8732

Std_2 10.000 10.599 6 2 987999 15876927 0.010 4.8716

Std_3 20.000 19.658 ^■2 3 1 61473 17615708 0.01 4.8825

Std_.4 62.500 60.153 -4 4 5563829 16808158 0.055 4.8734

Std_.5 50.000 48.987 -2 5 4721538 17459086 0.045 4.8716

Std_.6 100.000 96.775 -3 6 9592859 18106874 0.088 4,8713

Std_.7 250.000 244.797 -2 7 23979787 17971445 0.222 4,871 1

Std_8 400 000 2.508 3 8 40252034 7905472 0 375 4.8703

Std_9 500.000 509.298 2 9 49965048 17996378 0.463 4.8726

StdJO 750.000 732.247 -2 10 78874481 197366 2 0.666 4.8726

Std__ 11 000.000 1014.448 1 1 1 96247175 1 353278 0.924 4.8823

Sid 2 1250.000 1242.858 -1 12 128307307 18853095 1.134 4.8731

OC _.Low_._2 40.000 41.992 5 1 Low 775940 3337584 0.039 4.8924

OC _.Low_._2 40.000 33.880 -15 1 Low 743748 3945978 0.031 4.8930

QC_Low_1 40.000 38,644 -3 1 Low 3934913 18359852 0.036 4.8829

QC_Low_3 40.000 42,899 7 1 Low 775947 3268415 0.040 4.8917

QC_Med_1 200.000 89.461 -5 2 Med 9360578 873&738 0.172 4.8820

QC_Med_2 200.000 86.560 -7 2 Med 3045361 2992744 0.170 4.8825

QC_Med_2 200.000 201.031 1 2 Med 1686040 1537992 0.183 4.8938

QC__Med_._3 200.000 187.608 -6 2 Med 2096153 2048464 0.171 4.8928

QC__High__1 800.000 769.738 -4 3 High 78604327 18706868 0.700 4.8716

QC_. High_.2 800.000 705.358 -12 3 High 10431828 2710267 0.642 4.8840

QC_..High_..2 800.000 721.156 -10 3 High 5900132 1499181 0.656 4,8925

QC_High_3 800.000 8 9.576 2 3 High 0644697 2378535 0 746 4.8814

469 7 NA 14.854 NA NA 12137244 19328077 0.105 4,8730

46951 NA 142.801 NA NA 14998177 234280 0 130 4.8807

46960 NA 98.319 NA NA 0565017 631305 0 090 4.8829

46990 MA 28.847 NA NA 3132543 19434679 0.027 4.8920 47076 MA ■ 01.708 MA MA 11 42969 20020581 0.093 4.8835

47148 MA 59.043 MA MA 6308156 19409932 0.054 4.8838

47204 NA 02.845 NA NA 107 8957 19047492 0.094 4,8833

47223 NA 78.569 NA NA 8469906 19653465 0.072 4,8817

47259 NA 14.884 NA NA 12650349 20139926 0.105 4,8935

47262 NA 46.850 NA NA 5288079 20429903 0 043 4,8920

47271 NA 82.980 NA NA 8646221 190068 2 0.076 4.8834

47285 NA 54.159 NA NA 4680113 15679142 0.050 4.8833

47287 NA 36.896 NA NA 2820080 13767116 0.034 4.8823

47294 NA 84.735 NA NA 5185004 1 1 164296 0.077 4.8923

47296 NA 104,447 NA. NA. 10852415 18991199 0.095 4.8817

47362 NA 115,191 NA. NA. 4816356 7647588 0.105 4.8932

47398 NA 28,116 NA NA 905707 5760762 0.026 4.8929

47408 NA 102,226 NA NA 2846054 5087819 0.093 4.8923

47429 NA 56.922 MA MA 1821271 5809718 0.052 4.8843

47478 NA ■ 43.152 MA MA 3596365 4600874 0.130 4.8931

47507 MA 131.205 MA MA 2452209 3421 199 0.119 4.8923

47512 NA 85.263 NA NA 1482894 3173362 0.078 4,8935

47547 NA 221.439 NA NA 3947988 3270236 0.201 4,8910

47555 NA 72.270 NA NA 12 7277 3067942 0 066 4,8920

47583 NA 42.391 NA NA 594900 2535249 0 039 4,8922

47586 NA 36.423 NA NA 22 2374 2969176 0.124 4.8932

47626 NA 84.268 NA NA 14 7562 30690 7 0.077 4.8921

47629 NA 42.703 NA NA 531982 2250912 0.039 4.9043

47633 NA 80.594 NA NA 794839 1 98467 0.074 4.8826

47674 NA -0.750 NA. NA. 1647 2363729 0.000 4.9231

47696 NA ,835 NA NA 3332289 5087393 0.109 4.8923

477 5 NA 32,525 NA NA 1832445 253 225 0.121 4.9040

B109 MA 24.561 MA MA 393995 285632 0.023 4.9004

B 24 MA 87.045 MA MA 431391 904445 0.079 4.9035

B138 MA 97.370 MA MA 8771 1 1645556 0.089 4.9034

B151 MA 94.329 MA MA 715445 1385149 0.086 4.8937

B174 NA 56.169 NA NA 5 1797 1654158 0.052 4,8925

B209 NA 52.886 NA NA 1473081 1765109 0.139 4,8909

B2 1 NA 52.935 NA NA 329416 1128693 0 049 4,8927

B218 NA 77.648 NA NA 10390 9 24392 6 0.071 4.8930

B249 NA 91.092 NA NA 5975 0 1197560 0.083 4.8929

B348 NA 65.394 NA NA 867110 2412290 0.060 4.8935

B379 NA 149.254 NA NA 984468 1208202 0.136 4.8933

B4 6 NA 137,946 NA. NA. 873145 1158959 0.126 4.8937

B4 7 NA 119,721 NA. NA. 3160264 4829324 0.109 4.8927

B50 NA -0.596 NA NA 3075 20 0696 0.000 4.9146

B77 NA 65,086 NA NA 805769 2252079 0.060 4.8948

B793 MA ■ 02.335 MA MA 726520 1297409 0.093 4.8942

B875 MA 81.439 MA MA 1 83494 2650382 0.074 4.8927

B96 MA 60.352 MA MA 974518 2934438 0.055 4.8933

[8(5168] Patient data summary 4-OH- 4'-QH- N-DiW- N-DIW-4'-OH

Sample !D# Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen Endoxifen Norendoxifen

45301 130.875 1.778 4.590 26.273 3.852 19.943 5.074

45390 142.588 2.672 8.253 39.710 9.206 17.691 7.059

45466 51.861 0.965 3.246 14.131 3.310 15.559 4.693

45485 2.025 0.487 0.59 2.10 1.092 1.168 1.957

45589 -0.642 NA 0.019 -0.438 -0.222 HA 0.564

45634 79.422 0.873 3.033 18.333 2.705 14.921 3.811

45717 1 4.657 2.677 4.887 10.770 2.395 60.8 5 4.888

45750 26.715 0.612 2.340 4.916 2.489 5,976 3.368

45752 103.765 1.91 6.298 23.367 5. 30 12.995 5.275

45798 35.068 0.740 2.022 8.08 1.968 3.777 2.6

45800 50.405 1.669 5.486 12.381 6.013 12.760 7.030

45810 1 5.360 1.868 3.438 21.579 3.861 32.931 4.740

45825 173.854 1.142 6.583 33.817 5.314 20.421 4.487

45835 1 6.025 2.009 3.982 11.219 1.776 29.102 3.337

45867 90.584 1,537 3.108 12.834 2.016 29.304 4.885

45946 68.180 1.175 5.147 12.791 3.843 16.281 5.384

46037 122.733 2.761 5.643 10.380 2.595 59.095 4.910

46147 245.606 1.436 8.247 44.324 5.977 24.870 4.687

46153 64.050 1.205 3.098 13.248 2.827 17.213 3.27

46180 45.857 0.924 3.834 13.833 4.240 6.037 3.156

462 3 39.700 2.014 2.493 13.923 3.5 1 7.792 4.534

46221 60.817 1.115 3.541 26.106 5.312 16,512 5.436

46283 106.601 1.252 3.892 24.324 4.666 12.331 3.605

46301 175.670 1.076 5.251 29.768 5.016 9.789 2.748

46428 102.971 1.898 3.622 9.931 1.865 25.805 3.573

46442 34.971 0.620 2.922 6.573 2.995 6.342 3.827

46453 76.528 0.864 2.527 14.678 2.023 13.113 2.850

46434 134.492 1.441 3.743 15.352 2.667 30.740 4. 82

46518 32.897 0.721 3.108 5.940 3.264 8.837 6.699

46541 77.359 1.187 4.711 12.145 3.176 17.268 3.617

46548 85.387 1.205 4.970 17.369 5.486 7.076 3.513

46606 59.848 0.842 2.090 13.382 2.749 9.943 3.175

46667 135.598 5.743 10.785 54.075 16.979 23.458 12.284

46717 182.112 1.902 7.253 32.189 5.665 22.140 5.021

46731 145.855 0.965 4.273 23.463 5.254 9.207 3.454

46735 60.560 1.081 2.319 10.658 2.891 17.339 4.412

46749 97.135 1.764 3.876 18.278 3.681 25.845 4.007

46841 91.310 1.679 3.195 23.201 3.203 43.515 4.586

46859 129.621 3.643 6.391 35.736 7.708 21.258 8.215

46870 94.153 2.574 3.938 9.464 2.031 59.243 5.910

46896 152.754 1.602 6.647 39.389 7.929 9.876 5.884

46315 2.000 0.189 0.314 1.258 0.712 2.050 3.135

B1121 -0.601 0.014 -0.01 -0.449 -0.140 0.264 0.617

8423 140.264 2.679 7.737 33.052 5.703 42.457 1 .239

8593 68.107 0.976 5.042 5.587 4.554 6.927 2.742

8614 74.326 1.309 3.958 .595 3.239 26.725 6.182 B651 63.727 0.571 3.046 17.086 4.0S5 9.597 5.364

B702 67.581 1.861 3.499 20.529 4.310 24.278 7.953

4-OH- 4--OH- N-Di»- N-DM-4'-OH

Sample !D Tamoxifen Tamoxifers Tamoxifen Tamoxifers Tamoxifen Endoxifen Norendoxi

46317 114.854 1.781 6.294 26.132 5.430 10.963 3.918

46351 142.801 2.110 6,037 17,249 3.246 40.458 7,199

46980 98.319 0.564 4.332 18,233 3.288 9.167 4,594

46990 28.847 0 516 3.019 4,714 2.314 5.317 1 ,989

47076 01.708 1 806 4,647 7,044 2.692 28.357 4,324

47148 59.043 0.966 3.378 ■ 0.751 2.377 19.781 4.055

47204 102.845 0.580 4.003 21.609 3.480 11.477 6.512

47223 78.569 1.250 5.265 26.069 6.264 17.728 5.223

47259 1 14.884 0.793 4.429 24.482 4.635 14.089 3.709

47262 46.850 0.562 2.216 1.522 2.477 9.176 3.248

47271 82.980 1.230 3.533 6.032 3 110 20 053 3.1 7

47285 54. 59 0.606 2.812 0.523 2 169 8 497 2.922

47287 36.896 0.913 4.284 7.253 2.942 11.271 5.726

47294 84.735 1.479 6.148 23.672 4.107 28.117 8.31

47296 104.447 1.532 6.503 25.731 5.416 11.705 5.398

47362 115.191 1.41 6,575 30,720 5.126 22.343 4,939

47398 28.116 0.550 1.552 11 ,900 1.966 10.628 3,597

47408 102.226 2 128 4,236 14.802 2.134 38.171 3.988

47429 56.922 0 848 2.437 11 .723 2.483 20.228 7.582

47478 143.152 1.224 6.839 26.901 6.114 14.565 7.589

47507 131.205 0.642 5.091 25.265 4.416 7.048 3.479

47512 85.263 0.779 5.454 22.956 5.723 5.756 2.200

47547 221.439 1.777 8.056 39.779 6.325 22.285 5.877

47555 72.270 0.968 3.667 5.859 3.213 -j ·! 421 0.709

47583 42.391 1.163 6.473 8.888 3 863 10 629 7.439

47586 36.423 1.214 5.8 8 30.921 5 091 7 639 2.995

47626 84.268 0.511 3.746 8.322 3.172 8.915 9. 21

47629 42.703 0.374 3.136 5.135 2.413 5.126 2.355

47633 80.594 0.784 3.606 12.866 1.604 23.162 6.992

47674 -0.750 -0.071 0.093 -0.454 -0.234 0.223 2.439

47696 119.835 1.648 3,824 16,209 2.188 32.499 4,597

47715 132.525 1.562 5,750 13,470 2.948 24.293 4,958

B109 24.561 0 040 1.524 1 .432 2.366 .434 2,485

B 24 87.045 1 935 8.718 14.692 3.990 25.290 6.882

B 38 97.370 1.675 3.821 22.437 2.896 37.164 4.369

B151 94.329 0.461 4.594 21.416 3.940 7.651 3.439

B174 56.169 1.006 2.520 10.349 1.803 9.252 3.782

B203 152.886 1.928 6.388 14.006 2.088 33.909 33.557

B2 1 52.935 0.557 3.127 1.346 2.399 11.457 0.686

B2 8 77.648 .382 3.539 14.9 5 2 274 1 591 2.085

B249 91.092 0.929 5.207 5.747 2 646 18 802 1.890

B34S 65.394 0.420 3.459 5.742 2.069 10.935 1.837

8379 149.254 1.0 6 6.245 16.374 3.163 32.589 6.254

8416 137.946 1.436 3.862 13.889 1.218 26.686 0.898 B417 119.721 1.334 4.720 ■ 7.747 2.352 18.884 0.363

B50 -0.596 -0.104 0.111 -0.344 -0.128 NA 0.088

B77 65.086 0.589 4.677 13.2 3 4.478 4.298 NA

B793 102.335 1.034 5.094 6.843 3.572 17.569 1.393

B875 81.439 0.557 3.932 9.567 3.907 10.613 2.350

B96 60.352 0.437 2.546 9.888 2 496 14 510 5.071

[80169] The contents of the articles, patents, patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[8017(5] The meihods illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc, shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of descriptio and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present inv ention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the invention embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[80171] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within ihe generic disclosure also form part of the methods. This includes the generic description of the methods with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[80172] Other embodiments are within the following claims. In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

THAT WHICH IS CLAIMED IS:

1. A method for determining the amount of norendoxiien in a sample by mass spectrometry, comprising:

(a) ionizing said norendoxiien to produce one or more norendoxiien ions detectable by mass spectrometry;

(b) detecting the amount of the norendoxiien ion(s) from step by mass spectrometry; wherein the amount of the ion(s) detected is rel ated to the amount of norendoxiien in said sample.

2. The method oi claim 1, further comprising protein precipitation.

3. The method of claim 1, further comprising purification.

4. The method of claim 3, wherein said purification compri ses filtration.

5. The method of claim 3, wherein said purification comprises liquid chromatography.

6. The method of claim 5, wherein said liquid chromatography is high pressure liquid chromatography (HPLC) ,

7. The method of claim 1 , further comprising detecting the amount of an internal standard.

8. The method of claim 7, wherein said internal standard is a deuterated norendoxiien.

9. The method of claim 1, wherein said ionization is by atmospheric pressure chemical ionization (APCI).

10. The method of claim 1, wherein said ionization is in positive ion mode.

1 1. The method of claim 1, wherein said sample is a serum sample.

12. The method of claim 1, wherein said mass spectrometry is tandem mass spectrometry.

13. A method for determining the amount of tamoxifen and metabolites thereof in a sample in a single mass spectrometry assay, comprising:

(a) ionizing said tamoxifen and metabolites to produce one or more ions detectable by mass spectrometry;

(b) detecting the amount of the ion(s) from step by mass spectrometry; wherein the amount of the ion(s) detected is related to the amount of each of tamoxifen and meiaboiiies in said sample.

14. The method of claim 13, further comprising protein precipitation.

15. The method of claim 13, further comprising purification.

16. The method of claim 15, wherein said purification comprises filtration.

17. The method of claim 15, wherein said purification comprises liquid chromatography.

18. The method of claim 17, wherein said liquid chromatography is high pressure liquid chromatography (HPLC) .

19. The method of claim 13, further comprising detecting the amount of an internal standard.

20. The method of claim 19, wherein said internal standard is a deuterated internal standard.

21. The method of claim 13, wherem said ionization is by atmospheric pressure chemical ionization (APCI).

22. The method of claim 13, wherein said ionization is in positive ion mode.

23. The method of claim 13, wherein said sample is a serum sample.

24. The method of claim 13, wherein said mass spectrometry is tandem mass

spectrometry,

25. The method of claim 13, wherein said metabolites comprise norendoxifen.

26. The method of claim 13, wherein said metabolites comprise endoxifen or N- Desmethyl-4-Hydroxy Tamoxifen.

27. The method of claim 13, wherein said metabolites comprise 4 '-Hydroxy Tamoxifen.

28. The method of claim 13, wherein said metabolites comprise 4-Hydroxy Tamoxifen.

29. The method of claim 13, wherein said metabolites comprise N-Desmethyi-4'- Hydroxy Tamoxifen.

30. The method of claim 13, wherein said metabolites comprise N-Desmethyl Tamoxifen.

31. The method of claim 13, wherein said metabolites comprise norendoxifen, endoxifen, 4 '-Hydroxy Tamoxifen, 4-Hydroxy Tamoxifen, N-Desmethy 1-4 '-Hydroxy Tamoxifen, and N-Desmethyl-4 ' -Hydroxy Tamoxifen.